characterisation of genes encoding cyclophilins and …...2.14.2 total rna isolation 56 2.14.3...
TRANSCRIPT
i
Characterisation of genes encoding cyclophilins and
protein disulphide isomerases in cereals
Huimei Wu
(Master of Science)
This thesis is presented for the degree of Doctor of Philosophy
May 2010
Environment and Biotechnology Centre Faculty of Life and Social Sciences Swinburne University of Technology Melbourne, Australia
ii
Abstract
The storage protein quality of wheat is important for nutritional and food technological
purposes. ER-localised cyclophilins (Cyps) and protein disulphide isomerases (PDIs)
are expected to play crucial roles in the deposition of storage protein in cereals. Large
Cyp and PDI-like (PDIL) gene families have been identified in Arabidopsis and their
putative proteins show numerous potential functions. However, limited information
exists about the Cyp and PDI gene families in cereals.
This study aimed to identify and characterise the Cyp and PDI gene families in wheat
and rice using bioinformatics approaches and conduct comparative molecular and
phylogenetic analysis with their orthologues in Arabidopsis. Another main aim was to
experimentally identify and investigate the Cyp and PDI genes that potentially encode
endoplasmic reticulum-localised proteins.
Cyp family members (33 in rice, 22 in wheat) and PDIL family members (28 in rice, 27
in wheat) were identified using bioinformatics tools. In addition to the expected CypB
genes, ove, two more putative ER-localised Cyps were identified in wheat and these
may have potential roles in storage protein regulation. CypB genes were also isolated
experimentally from common wheat and its progenitors. The genomic and cDNA
sequences were cloned and the gene structure exhibited seven exons and six introns.
The putative ER-localised CypB protein translated from exon contig from the B genome
gene contains all PPIase signature sites, an N-terminal ER signal peptide, and an
atypical C-terminal ER localisation signal. The putative promoter sequences were
cloned and exhibit several potential tissue specific regulatory elements. The common
wheat CypB genes are localised to chromosomes 7AL, 7BL and 7DL.
In addition to the three reported ER-localised PDI homologues, five more putative ER-
localised PDIs were identified in wheat. Expression of three reported PDI homologues
in developing seeds as well as stems and leaves, supports the hypothesis that PDI plays
a role in vegetative tissues as well as in the developing endosperm.
iii
Collectively, the work has identified new Cyps and PDIs with potential roles in storage
protein folding function, response to ER and other stresses, grain quality and other
important traits.
iv
Acknowledgements
I am very grateful to my principal supervisor, Assoc. Prof. Mrinal Bhave, for her
enthusiasm, dedication and guidance during the planning and conduct of this project and
the preparation of this thesis. Her contribution of knowledge and dedication to the
project were remarkable and beyond my expectations. She gave me confidence,
inspired me and challenged me along the way.
I am very grateful to my co-supervisor, Prof. Russell Crawford for his support and
encouragement throughout the study.
I would like to extend my thanks to Prof. Rudi Appels at Murdoch University,
Australia, for his invaluable assistance and advice on the genetic mapping components
of my project.
In addition, many thanks to all of the PhD and Honours students in the lab, in particular
past and present members of Mrinal’s group (Kerrie, Emma, Mark, Pete, Paul, Abi and
Sheeping) for discussing problems and giving advice. I am grateful to the lab
technicians (Chris Key, Soula Mougos and Ngan Nguyen) for their constant willingness
to help.
I am very grateful to the Rural Industries Research and Development Corporation
(RIRDC) for providing me with financial support throughout this project with a Top-up
Postgraduate Scholarship.
The thesis was proofread by Campbell Aitken (Express Editing Writing and Research).
His editorial assistance improved grammar, punctuation and style; however, it did not
affect the structure, content or the analysis.
Thanks to my family, brothers and sister for their constant love and support. Special
thanks must go to my parents who have given me constant encouragement.
v
Finally, I want to express my deepest thanks and gratitude to my husband Zhonghua and
daughter Yilin for their unselfish love and support through my studies. I could not have
managed without them.
Huimei
vi
Publications arising from this work
Refereed journal article
Wu H, Wensley E, Bhave M (2009) Identification and analysis of genes encoding a
novel ER-localised Cyclophilin B in wheat potentially involved in storage protein
folding. Plant Science 176: 420-432.
Conference proceedings
Wu H, Bhave M (2009) A comparative analysis of the protein disulfide isomerase gene
family in rice and wheat. Proceedings of the 59th Australian Cereal Chemistry
Conference, 27-31 September, Wagga Wagga, Australia. (In press).
Wu H, Bhave M (2008) Molecular characterisation of Cyclophilin B genes and
promoter sequences in wheat and rice. Proc. of the 11th International Wheat Genetic
Symposium, 24-29 August, Brisbane, Australia. Sydney University Press. ISBN: 978-
1-920899-14-1. (Poster P220)
Wu H, Wensley E, Bhave M (2007) Molecular characterisation of Cyclophilin B genes
potentially involved in storage protein quality in wheat. Proceedings of the 57th
Australian Cereal Chemistry Conference, 5-9 August, Melbourne, Australia. (Eds.
Panozzo JF, Black CK). ISBN 1-876892-16-1. pp 230-233. (Poster P230).
vii
Declaration
I, Huimei Wu, declare that the PhD thesis entitled ‘Characterisation of genes encoding
cyclophilins and protein disulphide isomerases in cereals’ is no more than 100,000
words in length, exclusive of tables, figures, appendices, references and footnotes. This
thesis contains no material that has been submitted previously, in whole or in part, for
the award of any other academic degree or diploma, and has not been previously
published by another person. Except where otherwise indicated, this thesis is my own
work.
Huimei Wu
May 2010
viii
Abbreviations aa amino acid(s)
Aet Ae. tauschii
AFLP amplified fragment length polymorphism
APRL adenosine 5’-phosphosulfate reductase-like
AS allele-specific
At Arabidopsis thaliana
BAC bacterial artificial chromosome
BAR Botany Array Resource
BDT big dye terminator
BiP binding protein
BLAST basic local alignment search tool
bp base pair
CDS coding sequence
CN calcineurin
CS Chinese spring
CsA cyclosporin A
CT chloroplast/thylakoid
CTD C-terminal domain
Cyps cyclophilins
dATP deoxyadenosine triphosphate
dCTP deoxycytidine triphosphate
dGTP deoxyguanosine triphosphate
dH2O deionised water
DH doubled haploid
dTTP deoxythymidine triphosphate
dUTP deoxyuridine triphosphate
EDEM ER degradation-enhancing a-mannosidase-like protein
eFP electronic fluorescent pictograph
ER endoplasmic reticulum
ERAD ER-associated degradation protein
ERSE-II ER stress responsive element
esp2 endosperm storage protein 2
ix
EST expressed sequence tag
GAPDH glyceraldehyde-3-phosphate dehydrogenase
gDNA genomic DNA
Gm Glycine max (soybean)
GRXs glutaredoxins
GSDS gene structure display server
hCypA human CypA
HMW high molecular weight
Hsp heat shock protein
IPCR inverse PCR
kb kilobase pairs
kDa kilodaltons
LB Luria-Bertani medium
LZ leucine zipper
MEGA molecular evolutionary genetics analysis
MT mitochondria
MTS mitochondrial targeting signal
LS nuclear localization signals
NMR nuclear magnetic resonance
NT nullisomic-tetrasomic
N-terminal Amino terminal
Os Oryza sativa (rice)
PAC precursor-accumulating
PB protein body (bodies)
PCR the Polymerase Chain Reaction
PCD programmed cell death
PDI protein disulfide isomerase
PDILs protein disulphide isomerase-like proteins
Pin puroindoline
PM plasma membrane
PPIase peptidyl-prolyl cis-trans isomerase PPIase
PP2A protein phosphatase 2A
PRXs peroxi-redoxins
x
PSII photosystem II
PSV protein storage vacuole
QSOXL quiescin-sulfhydryl oxidase-like
QTL quantitative trait locus (loci)
RE restriction enzyme
RFLP restriction fragment length polymorphism
RNase ribonuclease
rpm revolutions per minute
RRM RNA recognition motif
RT-PCR reverse transcriptase PCR
SDS sodium dodecyl sulphate
SEF soybean embryo factor
SNP single nucleotide polymorphism
SP signal peptide
S-poor sulphur-poor
SQL structured query language
S-rich sulphur-rich
SSR simple sequence repeat
Ta Triticum aestivum (common wheat)
TAs Transcription assembles
TC Tentative consensus sequence
Td T. turgidum ssp. durum
TE Tris-EDTA buffer
TF transcription factor
TIGR The Institute for Genomic Research
TPR tetratricopeptide repeats
TRX thioredoxin
TSS transcription start site
Tu T. urartu
U units
UV Ultra-violet
wEST wheat EST
ZF zinc finger
xi
Table of contents
1 General introduction and literature review 2
1.1 Introduction to wheat as a crop 2
1.1.1 The wheat industry 2
1.1.2 Wheat flour composition and wheat quality 2
1.1.3 Wheat species and genomes 2
1.2 Wheat seed storage proteins: properties and significance 4
1.2.1 Classification and key biochemical properties of storage proteins 5
1.2.2 Genetic loci of storage proteins 6
1.2.3 Role of storage proteins in determining visco-elastic
properties of dough 7
1.2.4 Synthesis and deposition of seed storage proteins 8
1.2.5 Effects of abiotic stresses on storage proteins in wheat 11
1.2.6 Need for ‘foldase’ enzymes and chaperones in storage protein
folding and assortment processes 11
1.3 Cyclophilins 12
1.3.1 Discovery of cyclophilins 12
1.3.2 Cyclophilins as members of peptidyl prolyl cis/trans isomerases
superfamily 12
1.3.3 The primary biochemical activity of cyclophilins 13
1.3.4 Key structural and biochemical features of cyclophilins 14
1.3.5 Composition of the human cyclophilin family 15
1.3.6 Diverse functions of Cyp in humans and other organisms
(excluding plants) 16
1.4 Properties and functions of plant cyclophilins 18
1.4.1 Cyclophilin family in Arabidopsis: a summary 18
1.4.2 Properties and functions of Cyps in other plants 22
1.5 Protein disulfide isomerase (PDI) 24
1.5.1 Discovery of PDI 24
1.5.2 PDI as a member of the thioredoxin superfamily 25
1.5.3 Structure of PDI 26
1.5.4 The primary biochemical activity of PDI 27
1.5.5 Composition and function of human PDIL family members 29
xii
1.5.6 PDIL families in other organisms 31
1.5.7 Functions of PDI in human and other organisms 32
1.5.8 Additional functions of PDI relevant to cell biology and medicine 33
1.6 Properties and functions of plant PDIL 35
1.7 Evidence for roles of ER lumen ‘foldase’ enzymes and chaperones
in storage protein folding and deposition 39
1.8 Summary of the above literature and aims of this project 41
1.8.1 Aims of this project 42
2 Materials and Methods 45
2.1 Commercial kits, materials and solutions 45
2.2 Prepared solutions 45
2.3 Equipment 46
2.4 Plant materials and DNA stocks used in this project 47
General molecular methods
2.5 Growth of seedlings for DNA isolation 48
2.6 Preparation of genomic DNA 48
2.7 The polymerase chain reaction (PCR) 49
2.7.1 Synthesis of oligonucleotide primers 49
2.7.2 Typical PCR conditions 49
2.8 Agarose gel electrophoresis and purification of PCR products 51
2.9 Cloning of PCR products 51
2.9.1 Preparation of transformation-competent E. coli JM109 51
2.9.2 Cloning of genomic copies or cDNA of genes 51
2.10 Plasmid DNA isolation and RFLP analysis of polymorphism 52
2.11 DNA sequencing 53
2.11.1 DNA sequence reactions 53
2.11.2 DNA sequence analysis 53
2.12 Amplification of promoter sequences by inverse PCR (IPCR) 54
2.12.1 Promoter sequences amplification 54
2.12.2 Characterisation of amplified fragments 54
2.13 Physical and genetic mapping of wheat CypB genes 54
2.13.1 Physical mapping of wheat CypB genes 54
xiii
2.13.2 Genetic mapping of wheat CypB genes 55
2.14 RNA extractions and cDNA synthesis 56
2.14.1 Tissue samples 56
2.14.2 Total RNA isolation 56
2.14.3 First-strand cDNA synthesis 56
2.15 Preliminary analysis of some Cyps and PDILs genes expression 57
Bioinformatics Method 58
2.16 Identification of CypB gene and its promoter sequence in rice 58
2.17 Identification of putative wheat CypB orthologues at rice CypB locus 58
2.18 Identification of putative QTLs in rice and wheat at the CypB locus 59
2.19 Rice Cyp and PDIL family identification and analysis 59
2.20 Wheat Cyp and PDI family identification and analysis 59
2.21 Identification of putative QTLs related to quality in rice and
wheat at loci encoding PDILs 61
2.22 Multiple sequences alignments, cellular location predictions
and phylogenetic analysis 62
2.23 Prediction of PDIL expression in different tissues in Arabidopsis 62
3 Molecular characterisation of cyclophilin B genes in
wheat species and rice 64
3.1 Abstract 64
3.2 Introduction 65
3.3 Results 66
3.3.1 Amplification and identification of CypB genes from genomic
DNA and first-strand cDNA in T. aestivum 66
3.3.2 Sequence alignment of clones from genomic DNA and cDNA
in T. aestivum 68
3.3.3 Identification of two CypB genes in T. turgidum ssp. durum 76
3.3.4 Identification of CypB genes in T. urartu and tentative genome
assignments 77
3.3.5 Identification of CypB genes in Ae. tauschii and tentative
genome assignments 78
3.3.6 Intron/exon structures and sequence identities of various CypB genes 78
xiv
3.3.7 Alignment of exon contigs from genomic sequences
with cDNA clones 81
3.3.8 Alignment of the deduced putative CypB protein sequences
in wheat species 85
3.3.9 Comparison of putative CypB protein sequences with ER-localised
Cyps in other organisms and CypA in human and wheat 86
3.3.10 Identification of putative CypB gene and its promoter
sequence in rice 90
3.3.11 Identification of CypB promoter sequences in diploid wheat 91
3.4 Discussion 94
3.4.1 Wheat cyclophilin B genes show significant diversity in introns 94
3.4.2 Genome assignment of cyclophilin B genes supports the origins
of A genome and D genome from T. urartu and Ae. tauschii 95
3.4.3 Section(s) of Cyclophilin B genes may be divergent in D genome
of common wheat compared to its progenitor 95
3.4.4 Putative wheat CypB protein shows potential roles for
ER protein regulation 95
3.4.5 Wheat CypB has the orthologue in rice and other species 97
3.4.6 The promoter elements suggest CypB proteins may be under tissue
and stress specific regulation in cereals 97
4 Physical and genetic mapping of cyclophilin B genes 100
4.1 Abstract 100 4.2 Introduction 101
4.3 Results 101
4.3.1 Identification of putative wheat orthologues of the rice
cyclophilin B gene and their chromosomal positions 101
4.3.2 Development of molecular markers for physical mapping 102
4.3.3 Experimental physical mapping of wheat Cyp B genes 103
4.3.4 Confirmation of PCR product amplified with allele specific primers
from different genome in T. aestivum cv. Rosella 106
4.3.5 Identification of polymorphisms in TaCypB7A gene from
ten parental lines 107
xv
4.3.6 Identification of polymorphisms in TaCypB7B gene from
ten parental lines 108
4.3.7 Identification of polymorphisms in TaCypB7D gene from
ten parental lines 109
4.3.8 Genotyping of cyclophilin B markers in mapping population 109
4.3.9 Linkage mapping of TaCypB7A and TaCypB7B 114
4.3.10 Identification of rice and wheat QTLs at CypB location 119
4.4 Discussion 124
4.4.1 Data on physical mapping of wheat in rice database indicates
CypB genes may be duplicates on different chromosomes 124
4.4.2 Development of molecular markers for physical mapping
prior to designing of allele specific primers 124
4.4.3 Genetic mapping of TaCypB7A 125
4.4.4 Genetic mapping of TaCypB7B 125
4.4.5 Genetic mapping of TaCypB7D 125
4.4.6 QTLs in the chromosome regions of the cyclophilin B genes
in rice and wheat 126
5 A comparative phylogenetic and molecular analysis of the
cyclophilin family in rice and wheat 128
5.1 Abstract 128
5.2 Introduction 129
5.3 Results 131
5.3.1 Identification of genomic sequences of Cyps genes in rice 131
5.3.2 Identification of expressed sequences of Cyp genes in rice 131
5.3.3 Predictions of intron/exons structures of rice Cyp genes 132
5.3.4 Grouping of putative Cyp proteins in rice 134
5.3.5 Analysis of the rice Cyp gene structures 138
5.3.6 Cyp-coding DNA sequences in wheat 140
5.3.7 Comparative analyses of putative Cyp amino acid sequences
of rice and wheat 142
5.3.8 Chromosomal localisation of Cyps in rice 157
5.3.9 Prediction of chromosomal location of Cyps in wheat 158
xvi
5.3.10 Preliminary expression analysis of genes encoding putative
ER localized Cyps 160
5.4 Discussion 162
5.4.1 The Rice and wheat Cyps comprise large and divergent families,
with some duplicated genes 162
5.4.2 Single domain cytosolic Cyps 162
5.4.3 Multi-domain cytosolic Cyps 164
5.4.4 ER Cyps 165
5.4.5 Chloroplast Cyps 166
5.4.6 Nuclear Cyps 166
5.4.7 Mitochondrial Cyps 167
5.4.8 Chromosomal locations of Cyps in rice and wheat 167
6 A comparative molecular and phylogenetic analysis of the
protein disulphide isomerase (PDI) family in
rice and wheat 170
6.1 Abstract 170
6.2 Introduction 171
6.3 Results 172
6.3.1 Identification of genomic sequences of PDIL genes in rice 172
6.3.2 Identification of expressed sequences of PDIL genes in rice 173
6.3.3 Predictions of intron/exons structures of rice PDIL genes 173
6.3.4 Grouping of putative PDIL proteins in rice 173
6.3.5 Analysis of the rice PDIL gene structures 180
6.3.6 PDIL coding sequences in wheat 183
6.3.7 Comparison of the putative PDIL amino acid sequences of rice
and wheat to each other and to other plant PDILs 186
6.3.8 The relationship of PDILs in rice and wheat to those in Arabidopsis
and other organisms 199
6.3.9 Chromosomal localisation of PDILs in rice and prediction
of chromosomal location of PDILs in wheat 205
xvii
6.3.10 Identification of quantitative trait loci likely to be associated
with PDILs in rice and wheat 207
6.3.11 Expression analysis of selected PDILs in different tissues from
Arabidopsis microarray data 208
6.3.12 Preliminary expression analysis of three homeologous PDI genes
in different tissues and developing seeds in wheat 210
6.4 Discussion 212
6.4.1 Rice and wheat PDILs comprise large and diverse families 212
6.4.2 The PDIL1 group 212
6.4.3 The PDIL2 group 214
6.4.4 The PDIL5 group 215
6.4.5 The QSOXL group 215
6.4.6 The APRL group 216
6.4.7 Chromosomal locations of PDILs in rice and wheat 216
7 General discussion 219
7.1 General discussion 219
7.1.1 Cyclophilin B shows potential location in the ER 219
7.1.2 Putative ER localized cyclophilin and protein disulphide isomerases
may have roles in ER stress response 219
7.1.3 Plant cyclophilins in other cellular locations show
a variety of functions 221
7.2 Future directions 221
References 224
Appendices 251
xviii
List of Figures
Figure 1.1 Summary of the evolution of wheat 4
Figure 1.2 Classification of wheat seed storage proteins (prolamins) 5
Figure 1.3 Schematic summary of sequences of x-type and y-type HMW subunits 6
Figure 1.4 Head-to-tail arrangement of x- and y-type HMW glutenin subunits
with branches of LMW-glutenin 7
Figure 1.5 Processes of storage protein body (PB) formation 8
Figure 1.6 Schematic representation of RNA-dependent seed storage protein
targeting in developing rice endosperm 10
Figure 1.7 Composition of the peptidyl prolyl cis/trans isomerases (PPIase)
superfamily 13
Figure 1.8 Cis-trans isomerisation of a peptidyl-prolyl bond accelerated by
PPIase 13
Figure 1.9 Structure of the main domain and binding site of Cyp 14
Figure 1.10 Schematic representation of cyclophilin members in human 16
Figure 1.11 Structures of putative representative cyclophilins in Arabidopsis 19
Figure 1.12 Classification of the TRX superfamily 26
Figure 1.13 Domain organisation of human PDI 27
Figure 1.14 Disulfide formation and isomerisation accelerated by PDI 28
Figure 1.15 Mechanism of disulfide bond formation in native proteins 28
Figure 1.16 Schematic model illustrating the interaction between Ero1-L
and hPDI 29
Figure 1.17 Domains in human PDI members 30
Figure 2.1 Representative chart for identification of PDILs or Cyps
chromosomal locations in wheat 61
Figure 3.1 Position of primers used for PCR or IPCR amplification 66
Figure 3.2 CypB genomic gene sections and the RFLP polymorphisms
(HaeIII) of clones in T. aestivum, cv. Rosella and CypB cDNA
in T. aestivum, cv. Cranbrook 68
Figure 3.3 Alignment of sequence from seven CypB genomic genes in
wheat species 75
Figure 3.4 RFLP Polymorphisms (HaeIII) of CypB genomic clones in
tetraploid and diploid wheat 76
xix
Figure 3.5 Section amplified by inverse PCR in T. urartu (A) and
confirmation of clones with expected inserts (B) 77
Figure 3.6 Gene structures of the CypB genes isolated from wheat species
and the CypB gene identified in rice 79
Figure 3.7 Phylogenetic analysis of CypB exon contigs and cDNA sequences 82
Figure 3.8 Sequence alignment of exon contigs with cDNA sequences 84
Figure 3.9 Alignment of amino acid s deduced from exon contigs of the seven
CypB genomic genes and CypB cDNA of three clones 86
Figure 3.10 Comparison of ER-localised cyclophilins from different species
with CypA in human and wheat 90
Figure 3.11 Section CYPBIPCR-R1/CYPBF3 amplified by inverse PCR from
SacI digested and circularised genomic DNA (A) in Ae. tauschii
and confirmation of the clones with expected insertion (B) 92
Figure 4.1 Putative wheat orthologues at the CypB locus of rice 102
Figure 4.2 Deduced restriction maps of Exon III to Exon V sections of
TaCYPB-A, TaCYPB-B and TaCYPB-D genes from common wheat 103
Figure 4.3 Positions of primers for physical and genetic mapping of the
genomic CypB genes 104
Figure 4.4 Physical mapping of CypB genes using RFLPs 105
Figure 4.5 Physical mapping of three CypB genes from wheat 105
Figure 4.6 PCR products amplified with allele specific primers from different
genome of gDNA in T. aestivum, cv. Rosella and their RFLPs
digested by enzyme (HaeIII) 106
Figure 4.7 Partial CypB PCR products with allele specific primers from the A
genomes (A), B genome (B), D genome (C) in ten parental lines 107
Figure 4.8 Structure of partial CypB genes (partial Intron II to partial Exon V)
in Tasman and Sunco 107
Figure 4.9 Representative SNP in CypB genes from parental pair CD87 and
Katepwa)(A), alignment of sequences (867- 890bp) in parental lines
with TaCypB7B (B) 108
Figure 4.10 RFLPs of partial CypB gene from B genome in ten parental lines 109
Figure 4.11 TaCypB7A marker assay in the progeny from Tasman x Sunco 112
Figure 4.12 TaCypB7B marker assay in the progeny from Tasman x Sunco 114
Figure 4.13 Linkage map of chromosome 7B generated from Sunco x
xx
Tasman (A) and comparative linkage map of chromosome 7B
in T. aestivum (B) 116
Figure 4.14 Integration of TaCYPB7A and TaCYPB7B markers in the linkage
maps from Sunco x Tasman (Chalmers et al., 2001) based on
the bin mapped markers indicated in Section 4.3.1 (Fig 4.5)
and data analysis by Map Manager software package
QTXb20 (Fig 4.13A) 118
Figure 5.1 Gene structures of Cyp gene family in rice 133
Figure 5.2 Predicted cellular locations of cyclophilins in wheat and rice
compared to those reported in Arabidopsis 135
Figure 5.3 Structure of putative Cyp proteins in rice 137
Figure 5.4 Structure of putative Cyps proteins in wheat 142
Figure 5.5 Alignment of putative amino acid sequences of rice and wheat Cyps
with reported Cyps in Arabidopsis and human CypA 154
Figure 5.6 Phylogenetic analysis of Cyps in Arabidopsis, rice, and wheat 157
Figure 5.7 Likely locations of rice Cyp genes on chromosomes 158
Figure 5.8 Summary of likely locations of wheat Cyps on chromosomes 159
Figure 5.9 RT-PCR products amplified from the first-strand cDNA or genomic
DNA of various tissues and developing seeds of wheat 161
Figure 6.1 Rice PDIL gene structures 174
Figure 6.2 Schematic of the structures of PDILs in rice 178
Figure 6.3 Schematic of the structures of PDILs in wheat 185
Figure 6.4 Alignment of the putative amino acid sequences of the five groups
of PDILs in rice and wheat 198
Figure 6.5 Phylogenetic analysis of PDILs in Arabidopsis, rice, and wheat 203
Figure 6.6 Likely Locations of rice PDILs on chromosomes 205
Figure 6.7 Likely chromosomal locations of wheat PDILs 207
Figure 6.8 PDIL expression levels in different tissues in Arabidopsis (upper)
and expression of AtPDIL1-1 in developing seeds lower 209
Figure 6.9 RT-PCR products amplified from the first-strand cDNA or genomic
DNA of various tissues and developing seeds of wheat 211
xxi
List of Tables
Table 1.1 Key properties and functions of cyclophilins 17
Table 1.2 Properties of cyclophilin family in Arabidopsis and their orthologues 21
Table 1.3 Key properties of cyclophilins in other plants 24
Table 1.4 Properties and functions of PDI in vertebrates and yeast 34
Table 1.5 Properties and functions of PDIL members in Arabidopsis and their
orthologues in other plants 36
Table 1.6: PDI properties and functions in plants (excluding Arabidopsis) 39
Table 2.1 Commercial kit and reagents used during this project 45
Table 2.2 Prepared solutions, media and plates 46
Table 2.3 Equipment used during this project 45
Table 2.4 Wheat cultivars and progenitors 47
Table 2.5 Nullisomic/tetrasomic (NT) lines used for physical mapping 47
Table 2.6 Deletion lines of chromosome 7 used for physical mapping 48
Table 2.7 Primer pairs used for PCR or IPCR amplifications and/or sequencing
of CypB gene and its promoter, physical mapping and genetic mapping 50
Table 2.8 Primers used for gene expression study 57
Table 3.1 Primer pairs used for PCR or IPCR amplification/or sequencing of
CypB gene and its promoter 66
Table 3.2 Summary of sequenced CypB gene sections in wheat species 69
Table 3.3 Identities amongst the wheat CypB genes and with rice and
Arabidopsis orthologs 80
Table 3.4 Sequences of exon/intron junction and putative intron branch points
in TaCypB7B 81
Table 3.5 Functionally important residues in the putative wheat and
rice CypB proteins 90
Table 3.6 Sequence comparisons of the isolated TaCypB7B gene and its
putative protein product, with those from rice 91
Table 3.7 Putative regulatory elements in the promoters of CypB genes
in wheat and rice 94
Table 4.1 Primer pairs used for physical and genetic mapping 104
Table 4.2 QTLs in the vicinity of the rice CypB locus on chromosome 6 120
xxii
Table 4.3 Markers of QTLs controlling grain or protein quality on rice
chromosome 6, in the general area of the CypB gene 121
Table 4.4 Wheat QTLs relating to agronomic and quality traits on chromosome 7 122 Table 4.5 Summary of wheat QTLs for agronomic and quality traits
and their closest markers on chromosome 7 123
Table 5.1 List of Rice Cyclophilin gene family 134
Table 5.2 Summary of exon sizes (bp) of Cyp genes in rice 139
Table 5.3 Cyclophilin family in Triticum aestivum 141
Table 5.4 Reported functionally important residues in human CypA 143
Table 5.5 Primers used for expression analysis of ER-localising Cyp genes 160
Table 6.1 PDIL gene family in rice 175
Table 6.2 Identities (%) between putative amino acid sequences and
between the coding sequences of PDILs in rice 179
Table 6.3 Summary of exon sizes (bp) of PDIL genes in rice 182
Table 6.4 PDIL family in wheat 186
Table 6.5 PDIL members in wheat and their potential orthologues
in other plants 204
Table 6.6 Wheat and rice putative PDIL orthologues and their chromosomal
locations 206
1
Chapter 1
GENERAL INTRODUCTION AND LITERATURE REVIEW
2
1 General introduction and literature review
1.1 Introduction to wheat as a crop
1.1.1 The wheat industry
Wheat is a staple food crop grown in many different countries. The wheat growing
areas total approximately 204,614,529 hectare (ha) worldwide (NUE, 2003), and about
620 million tons are produced annually (Dubcovsky and Dvorak, 2007). Wheat is
Australia’s largest crop, occupying approximately 12,456,000 ha in 2003 (NUE, 2003).
In 2006/2007, the Australian wheat yield was 10.8 million tons; 8.7 million tons with a
value of $3 billion were exported (ABARE, 2008a). Global wheat stocks are currently
low due to demand exceeding production (ABARE, 2008b), and improvements in
global wheat yield are urgently needed. Wheat flour is the most important ingredient of
bread, pasta, noodles and a range of staple foods, so is thus essential to keep this crop
abundant and affordable and improve its quality.
1.1.2 Wheat flour composition and wheat quality
Wheat flour is processed by milling to remove the germ (embryo) and bran. The main
part consumed by humans is the endosperm, which typically contains 72% starch
(Pomeranz, 1988) and 10-12% storage proteins, the quality of grain being dependent on
the characteristics of these polymers (reviewed in Shewry and Halford, 2002). The
gluten proteins form a continuous matrix in the mature dry endosperm cells in wheat.
Flour is mixed with water to form dough, which is expanded by fermentation and baked
into leavened bread or processed into pasta, noodles and a range of other foods. The
technological properties of starch are determined by many factors including its amylose
content, branched chain-length distribution of amylopectin, and starch granule size
distribution (Gama et al., 2000). Similarly, the storage proteins (‘gluten’) show clear
associations with nutritional quality and the visco-elastic properties of the dough due to
properties of individual subunits and overall structure of the polymer; these are detailed
later.
1.1.3 Wheat species and genomes
Domestication of wheat is generally accepted to have occurred around ~12,000 years
ago in the Fertile Crescent of western Asia, most likely in southeastern Turkey. Wheat
spread through Iran into central Asia reaching China by about 3000 BC, and was
3
introduced in Australia in 1788. The origin and domestication of wheat and the original
research reports on this have been reviewed extensively elsewhere (Salamini et al.,
2002; Huang et al., 2002; Dubcovskyi and Dvorak, 2007; Shewry, 2009). Wheat is a
member of the tribe Triticeae, a subdivision of Panicoideae subfamily belonging to the
Poaceae (grass) family. Based on a basic set of 7 chromosomes (n=7), all wheat
species are divided into diploid (2n=2x=14), tetraploid (2n=4x=28) or hexaploid
(2n=6x=42) (Breiman and Graur, 1995). The diploid species include three genome
types. The AA genome type is represented mainly by Triticum urartu and T.
monococcum (Sears, 1969), the BB genome is represented by various species including
Agropyron triticeum, Aegilops bicornis and Ae. speltoides, while the DD genome is
represented by Ae. tauschii (reviewed in Kimber and Athwal, 1972). The tetraploid
wheats fall into two genome types; the AABB includes T. turgidum which has two sub-
species: the wild form (T. turgidum ssp. dicoccoides) and the cultivated form (T.
turgidum ssp. durum) (reviewed in Morris and Sears, 1967), while the AAGG type has
only one species, T. timopheevii, with two sub-species, the wild T. timopheevii ssp.
armeniacum and the cultivated T. timopheevii ssp. timopheevii (reviewed in Breiman
and Graur, 1995). The hexaploid wheats include two genome types; the AmAmAAGG
is represented by one species (T. zhukovskyi) while AABBDD includes one species (T.
aestivum L.) with sub-species T. aestivum ssp. vulgare (bread wheat), T. aestivum ssp.
spelta (spelt wheat), T. aestivum ssp. compactum, T. aestivum ssp. macha, T. aestivum
ssp. vavilovii, and T. aestivum ssp. sphearococcum (club wheat).
The generally accepted theory of evolution of common wheat is shown in Fig 1.1. Ae.
speltoides (BB, 2n=14 chromosomes) is proposed to have hybridised with T. urartu
(AA, 2n=14) and chromosomes of the AB hybrid may have doubled to produce the
tetraploid T. turgidum (AABB, 4n=28). T. turgidum then possibly hybridised with Ae.
tauschii (DD, 2n=14) and chromosomes of this 3n, ABD hybrid also probably doubled,
leading to the common wheat (T. aestivum, AABBDD, 2n=42). Cytogenetic analysis
shows the A genome was probably contributed by T. urartu; the B genome by Ae.
speltoides (Sear, 1969; Breiman and Graur, 1995); and the D genome by Ae. tauschii
(Gill and Kimber, 1974). Molecular evidence for the A genome donor being T. urartu
includes comparison of 18S.26S ribosomal RNA loci (Nor-A7 on 5AL) by sequential
N-banding and in situ hybridisation (Jiang and Gill, 1994), RFLP comparison of RbcS
gene (Galili et al., 2000), and 5S spacer rDNA from homeologous chromosome 1
4
(Allaby and Brown, 2000). The B genome being from Ae speltoides is supported by
similar analyses of 18S·26S rRNA loci (Nor-B6 on 1BL) (Jiang and Gill, 1994) and
RbcS (Galili et al., 2000), or it may have come from an unknown relative of Ae
speltoides (Feuillet et al., 2008). The D genome donor status of Ae. tauschii is also
confirmed by analysis of 18S·26S rRNA loci (Nor-D8 on 3DS) (Jiang and Gill, 1994)
and the rRNA (Nor3), high molecular weight glutenin (Glu1) and RbcS loci (Dvorak et
al., 1998; Galili et al., 2000).
Fig 1.1 Summary of the evolution of wheat (reviewed in Shewry, 2009). 1.2 Wheat seed storage proteins: properties and significance
1.2.1 Classification and key biochemical properties of storage proteins
The wheat storage proteins (‘gluten’) were one of the earliest plant proteins to be
analysed. Historically, they were divided into four groups according to extraction and
solubility characteristics, i.e., albumins (soluble and extractable in water and dilute
buffers), globulins (not soluble in water but soluble and extractable in dilute saline),
gliadins (soluble and extractable in 70-90% ethanol) and glutelins (soluble and
extractable in dilute acid or alkali; these were later divided into two separate classes)
(reviewed in Gianbelli et al., 2001). All wheat storage seed proteins are generically
termed prolamins, due to being rich in proline and glutamine. The history of
classification of wheat prolamins has been covered extensively elsewhere (Shewry et
5
al., 1995; Gianibelli et al., 2001). As per the commonly accepted theme (followed
here), the prolamins are divided into two groups based on degree of polymerisation: the
monomeric gliadins and polymeric glutenins. Based on amino acid composition, they
fall into three broad groups: S-rich (sulphur-rich), S-poor, and high molecular weight
(HMW) (Shewry and Halford, 2002), and the two classification systems can be
integrated (Fig 1.2). The S-poor prolamins have a partial composition of 40-50% Gln,
20-30% Pro, 8-9% Phe, 0-0.5% Lys and 0-<0.5% Cys and contain monomeric ω-
gliadins and the D-type low molecular weight (LMW) glutenin subunits that can form
polymers. The S-rich proteins comprise the main component (70-80%) of prolamins,
have a partial amino acid composition of 30-40% Gln, 15-20% Pro, 2-3% Cys and
<1.0% Lys, and include three subgroups: the monomeric α-gliadins and γ-gliadins, and
the polymers of B- and C-type LMW glutenin subunits. The HMW prolamins have a
partial composition of 30-35% Gln, 10-16% Pro, 15-20% Gly, 0.5-1.5% Cys and 0.7-
1.4% Lys and form polymers of HMW glutenins (Shewry and Halford, 2002). The B-
and C-type subunits contain six or eight Cys, respectively, and have about 70
components (Muccilli et al., 2005).
Fig 1.2 Classification of wheat seed storage proteins (prolamins) (Shewry et al. 2003).
As the name suggests, the S-poor prolamins lack Cys residues and thus cannot form
disulfides bonds and polymers. The amino acid sequence of ω-gliadins contains only
one domain: the repeated motif (Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln), flanked on N- and
C-terminal sides by unique 12- and 9-residue sequences, respectively (Shewry and
Halford, 2002). The S-rich prolamins contain two main domains: the Pro/Gln-rich
6
repeated sequence (e.g., PQQPFQQ in γ-gliadin) toward the N-terminal, and the non-
repeated C-terminal which has Cys that form intra-chain disulfide bonds. Six Cys occur
in α-gliadins and eight in γ-gliadins (Shewry et al., 1995). The HMW glutenin subunits
contain three distinct domains: the central domain (420-700 residues) with extensive
repeats comprised mainly of nonapeptide and hexapeptide motifs, and often another
tripeptide; and the non-repetitive N-terminal (81-104 residue) and C-terminal (42
residue) domains which contain Cys that form inter-chain disulfides (Shewry et al.,
2002) (Fig 1.3). The HMW subunits fall into two types: the x-type has an additional
tripeptide motif in the repetitive part and three Cys in the 81-89 residue N-terminal
domain, while the y-type has five Cys in the 104 residue N-terminal. In particular,
1Dx5 contains a Cys towards the N-terminal end of the repetitive domain, while 1By
and 1Dy types contain a Cys toward its C-terminus. The variations in sequences and
numbers of the repeats and Cys residues of HMW glutenins thus have major
implications for structure of the gluten polymer and its technological characteristics
(reviewed in Shewry et al., 1995; 2002).
Fig 1.3 Schematic summary of sequences of x-type and y-type HMW subunits (Shewry et al., 2002). Information in boxes indicates numbers of total residues and Cys in various domains; two N-terminal Cys are deleted in x-type; 1Dx5 contains one Cys close to the N-terminus of the repetitive domain; and 1By and 1Dy contain one Cys close to C-terminus of this domain.
1.2.2 Genetic loci of storage proteins
The Gli-2 loci encoding α-gliadins are located on short arms of chromosomes 6A, 6B,
and 6D, and the Gli-l loci controlling γ- and ω-gliadins are on short arms of
chromosomes 1A, 1B, and 1D. The Glu-I loci encoding HMW glutenin subunits occur
on long arms of chromosome 1A, 1B, and 1D, each locus having two genes, encoding
the x- and y-type subunits. Thus, theoretically, T. aestivum could contain six HMW
subunits (1Ax, 1Ay, 1Bx, 1By, 1Dx, 1Dy); however, silencing certain homeoalleles
7
results in the presence of only three (1Bx, 1Dx, 1Dy) to five (1Ax, 1Bx, 1By, 1Dx,
1Dy) subunits (Payne, 1987).
1.2.3 Role of storage proteins in determining visco-elastic properties of dough
The visco-elastic properties of dough, formed by mixing flour with water, are very
important to the making of bread, pasta, noodles and many other foods (reviewed in
Payne, 1987). The structures of the HMW subunits influence these properties, as
demonstrated by the transgenetic line of wheat containing additional 1Dx5 subunits
(Barro et al., 1997). Polymeric prolamins are composed of the HMW x- and y-type and
LMW subunits, connected by intra- and/or inter-molecular disulfide bonds. The HMW
subunits form the backbone of a head-to-tail structure from N- to C-terminus, while the
LMW subunits form branches, all connected via disulphide bonds (Fig. 1.4). One
interchain disulphide bond is present at the N-terminus of x-type subunits and two
parallel disulphides occur between N-termini of y-type subunits (Shewry et al. 2002).
The HMW glutenin stabilised by intramolecular disulphide bonds and inter-molecular
hydrogen bonding contribute to dough viscosity, while LMW glutenin stabilised largely
by inter-molecular disulphides determine dough elasticity and strength (reviewed in
Shewry et al., 2002; 2003). Transgenic lines were used to study the effects of the 1Ax1
and 1Dx5 subunits on the visco-elastic properties of the dough; the expression of 1Ax1
transgene resulted in a substantial increase in and time taken to mix to the maximum
resistance of dough, while expression of 1Dx5 resulted in increases in elasticity
(reviewed in Shewry et al., 2002).
Fig 1.4 Head-to-tail arrangement of x- and y-type HMW glutenin subunits with branches of LMW-glutenin (Shewry et al., 2002).
8
1.2.4 Synthesis and deposition of seed storage proteins
Seed storage protein synthesis and deposition in the endoplasmic reticulum (ER) was
first studied in the maize endosperm by electron microscopy (Larkins and Hurkman,
1987). The proteins may remain in the ER or be transported to distal sites through the
endo-membrane system (Herman and Larkins, 1999). It is now widely accepted that the
overall process involves three key steps: (i) synthesis of protein subunits on ribosomes;
(ii) targeting of nascent proteins to the lumen of ER where their folding and assembly
takes place; (iii) assembly of mature proteins into protein bodies (PBs) and deposition
of PBs in the endosperm (reviewed in Herman and Schmidt, 2004; Vitale and Ceriotti,
2004; Hwang, 2008) (Fig 1.5A). The nascent protein subunits have an N-terminal
signal peptide for directing them into the ER lumen, which is then cleaved (reviewed in
Shewry, 1999). The enzymes and molecular chaperones in the ER lumen, e.g., protein
disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase (PPIase) and Binding
Protein (BiP) possibly assist in the folding of nascent subunits, formation of disulfide
bonds and assembly of oligomers (see below). Misfolded nascent proteins are regulated
to either reform correct conformation or degraded with the aid of chaperones (Meunier
et al., 2002; Vitale and Ceriotti, 2004) (Fig 1.5B).
Fig 1.5 Processes of storage protein body (PB) formation (Vitale and Ceriotti, 2004). A: Nascent subunits folded and PBs assembled with the aid of chaperones. B: Misfolded nascent subunits subjected to aggregation for assembling into PBs, or degradation with the aid of chaperones.
The gliadins were initially identified by immunocytochemical techniques to be localised
in vesicles associated with the Golgi apparatus in wheat endosperm (Kim et al., 1988).
9
PBs were also detected in the cytoplasm by ultrastructural analysis, showing a dense
core surrounded by rough ER-derived membranes and probably enlarging by fusion of
smaller PB (Levanony et al., 1992). Two complementary pathways are now widely
accepted to lead to PB formation in wheat: some proteins (primarily gliadins) follow the
standard secretory pathway via Golgi to the protein storage vacuole, where they
accumulate into PBs, while the HMW glutenins accumulate directly into PBs within the
lumen of rough ER (reviewed in Shewry and Halford, 2002; Shewry et al., 2002;
Herman and Schmidt, 2004, Tosi et al., 2009), although the movement and localisation
of LMW subunits of glutenins is unclear (Tosi et al., 2009), and assortment of specific
prolamin types into two different PBs has been disputed (Loussert et al., 2008).
Curiously, all prolamins lack the archetypical KDEL/HDEL ER-retention signal or a
discernable vacuolar targeting signal, hence it is also unclear why some prolamins
follow the Golgi pathway to vacuole while others are retained in the ER. The proline-
rich repeats appear to be essential for ER retention (Altschuler et al., 1993; Altschuler
and Galili, 1994), possibly due to a role in protein-protein interactions that result in
accumulation in ER, making them less readily transportable through Golgi as compared
to the monomeric gliadins (Shewry and Halford, 2002). The ER-derived PBs appear to
disconnect from the cisternal ER, surrounded by ER membranes, but the process of
budding remains unknown (reviewed in Galili, 2004). The ER-derived PBs may
ultimately fuse with PBs of vascular origin (reviewed in Galili et al., 1997; Tosi et al.,
2009). The synthesis of large amounts of protein appears to be regarded as an internal
stress, and in response, the ER differentiates into various compartments with different
mechanisms of origin, shapes and functions (reviewed in Hara-Nishimura et al., 2004).
The PBs from Golgi are sequestered in protein storage vacuoles by a process similar to
autophagy (Vitale and Galili 2001). The membranes lyse and PBs coalesce, resulting in
a large central vacuole with one-more prolamin accretions towards the end of seed
maturation. However, details of PB fusion processes are unknown in wheat (Arcalis et
al., 2004) and other cereals (Galili, 2004; Herman and Schmidt, 2004). The SNARE
(soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein
(VTI12) may mediate the transport of PBs by the pathway via Golgi to PSV, as shown
by studies of mutant lines in Arabidopsis (Sanmartin et al., 2007). The eventual fusion
of all PBs in the endosperm forms the highly structured protein network (gluten) that
surrounds the starch granules and materials of broken-up endosperm cells and
determines the strength and viscoelastic properties of the dough. Factors that influence
10
gluten architecture thus potentially regulate the end-uses of wheat and are the subject of
extensive plant breeding efforts for nutritional as well as food technological reasons
(reviewed in Shewry and Halford, 2002; Shewry, 2009).
In the developing grains of rice, two types of PBs occur: the spherical PB-I contains
prolamins originating from ER, and the irregularly shaped PB-II containing globulins/
glutelins originating from the vacuole (Fig 1.6). Importantly, the prolamin and glutenin
mRNAs appear to be directed to specific subdomains of ER, i.e., protein body ER (PB-
ER) and cisternal ER (C-ER), respectively, and the 3’ untranslated regions on mRNAs
may play a role in targeting (Crofts et al., 2004). Translation of prolamin mRNAs on
PB-ER leads to translocation of the polypeptides into ER lumen and formation of PB-I
directly within ER. In contrast, partial prolamin proteins passing through a precursor-
accumulating (PAC) vesicle-dependent pathway, and glutelins translated from mRNAs
on C-ER and transported through Golgi, together get deposited as PB-II into a protein
storage vacuole (PSV) (Crofts et al., 2004).
Fig 1.6 Schematic representation of RNA-dependent seed storage protein targeting in developing rice endosperm (Crofts et al., 2004). Prolamin and glutelin mRNAs are transcribed in the nucleus (N) and transported into PB-ER and C-ER, respectively. Prolamin polypeptides are translocated into the ER lumen where they form PB-I, while glutelins are exported via the Golgi and deposited into PB-II.
11
1.2.5 Effects of abiotic stresses on storage proteins in wheat
The effects of environmental factors such as drought, salinity, cold, frost, water logging,
nitrogen, phosphorus and heavy metals, and unseasonable changes to normal patterns,
have been thoroughly studied and the literature is extensive (e.g., Morison et al., 1999;
Wheeler et al., 2000; Reddy et al., 2004). Recent work by Semenov and Halford (2009)
offers in-depth discussion on predictions and simulations of future climate changes and
roles of biotechnology in elucidating and addressing the emerging adversities. An
extensive discussion on abiotic stresses is outside the scope of this work, however, in
summary, it can be said that the results almost always point to severe effects on multiple
facets of plant growth including root function, water availability and stomatal function,
and significant losses of crops including wheat. For example, heat stress influences the
formation of polymeric protein fraction during grain filling in wheat (Ciaffi et al.,
1996). The total protein content is also affected by heat and drought stress through
altering the quantity of total N accumulation (Triboi et al., 2003). High temperature can
increase the rate for accumulation of S-poor compared to S-rich proteins (Dupont and
Altenbach, 2003; Dupont et al., 2006) and has been found to upregulate 31 proteins with
functions in stress/defense and storage and downregulate 32 proteins with roles in starch
biosynthesis and protein synthesis (Hurkman et al., 2009). The decreased proteins
include protein disulfide isomerase (PDI; also a chaperone) which catalyzes disulfide
bond breakage and formation in nascent proteins (Creighton et al., 1980; Hurkman et
al., 2009); PDI is a focus of this work and will be discussed below.
1.2.6 Need for ‘foldase’ enzymes and chaperones in storage protein folding and
assortment processes
The processes of mature storage protein folding, assortment and deposition occur in the
ER, as summarised above. During protein folding, incorrect disulphide bonding or
associations of hydrophobic residues can form undesirable interactions within or
between polypeptide chains, resulting in protein aggregates that need to be resolved or
targeted for removal. Not only do the normal events of rapid expression and assortment
of seed storage proteins lead to an internal ER stress (see section 1.2.4), external abiotic
stresses can affect physiological processes, as mentioned above. This supports the need
for an involvement of ‘foldase’ enzymes with specific biochemical properties as well as
chaperone roles, and other chaperones, for proper folding of nascent proteins and
resolution or removal of aggregates. Three groups of proteins are likely to be involved:
12
the Binding Protein (BiP)/heat shock protein (Hsp70) chaperones, and the enzymes
peptidyl prolyl cis-trans isomerases (PPIases) and protein disulphide isomerases (PDI).
The cyclophilins (a class of PPIases) and PDI are of direct relevance to this project.
Their key biochemical properties as well as experimental and circumstantial evidence
related to their roles in cereal storage protein folding and assortment processes are
detailed below.
1.3 Cyclophilins
1.3.1 Discovery of cyclophilins
Cyclophilins are so-named due to being cellular receptors for the immuno- suppressant
drug cyclosporin A (CsA) (Handschumacher et al., 1984). Cyclophilins (henceforth
designated as Cyps; literature shows use of this term, as well as ‘cyp’ or ‘CyP’) were
initially found to catalyse the cis-trans inter-conversion of proline (Pro)-containing
peptides purified from pig kidney (Fish et al., 1984). Early Cyps were reported from
different cellular locations, e.g., CypA from cytosol (Harding et al., 1986), CypB in the
ER (Price et al., 1991; 1994), and a nuclear-localized Cyp (Mortillaro and Berezney,
1998). Eighteen Cyps, ranging from 18-to 354 kDa, have been identified from human
genome (Galat, 2004). Cyps have also been reported from bacteria (Herrler et al.,
1994), yeast (Haendler et al., 1989), protozoa (Barisic et al., 1991) and plants (Luan et
al., 1994b, He et al., 2004; Romano et al., 2004a).
1.3.2 Cyclophilins as members of peptidyl prolyl cis-trans isomerase superfamily
Members of the peptidyl prolyl cis/trans isomerase (PPIase) superfamily catalyse the
cis/trans isomerisation of the peptide bond preceding Pro residues. Three proteins
families are reported to have this activity: (i) immunophilins that bind immuno-
suppressive drugs (Galat, 1993); (ii) the very low molecular weight (10.1 kDa)
parvulins (Landrieu et al., 2000); (iii) the ‘trigger factors’ required for translocation-
competent folding of pro-OmpA and other precursor proteins (Crooke and Wickner,
1987). Immunophilins are further divided into two subfamilies: cyclophilins that bind
to CsA (Handschumacher et al., 1984; Galat, 1993), and FKBPs that bind to FK506 and
rapamycin (Harding et al., 1989; Galat, 2003) (Fig 1.7).
13
Fig 1.7 Composition of the peptidyl prolyl cis/trans isomerases (PPIase) superfamily.
1.3.3 The primary biochemical activity of cyclophilins
The cis/trans isomerization of the peptide bond preceding Pro residues (Xaa-Pro) is a
rate-determining step in nascent protein folding and refolding. The main function of
Cyps in vertebrates was found to be the catalysis of this slow event (Fischer et al., 1984)
(Fig 1.8), this activity being inhibited by CsA in regulation of T-lymphocyte activity
(Handschumacher et al., 1984). The nature of this step was exemplified through studies
on isomerisation of the bond between Tyr-92 and Pro-93 in bovine pancreatic
ribonuclease A (Schmid et al., 1986). The 21 kDa Cyp in the ER lumen was shown to
interact with secretory proteins translocated into the lumen (Klappa et al., 1995). The
PPIase activity of Cyp is inactive in its oxidised form and regained by thioredoxin
converting it to its reduced form (Motohashi et al., 2001; 2003).
Fig 1.8 Cis-trans isomerisation of a peptidyl-prolyl bond accelerated by PPIase (Galat, 1993).
14
1.3.4 Key structural and biochemical features of cyclophilins
Individual Cyps range from 163 amino acids (aa) (~17kDa) in vertebrates (Harding et
al., 1986) to 837 aa (~95kDa) in plants (Lorkovi et al., 2004). All Cyps contain the
cyclophilin-like domain (CLD) (approximately 109 aa), connected to other domains that
are unique to each member and associated with subcellular location or functional
specialisation (Wang and Heitman, 2005). The structure exhibits eight-stranded
antiparallel β-barrels and two α-helices (Kallen et al., 1991). Based on the sequence of
hCyPA (Ke et al., 1991) these are as follows: (i) β-strands (B1: Pro4-Ala11, B2: Pro16-
Leu24, B3: Lys49-Ile56, B4: Met61-Gly64, B5: Gly96-Ala101, B6: Gln111-Thr116,
B7: Val127-Glu134, B8: Lys155-Leu164); (ii) β-turns (T1: Val12-Glu15, T2: Gly42-
Gly45, T3: Ile57-Phe60, T4: Ser77-Gly80, T5: Gly104-Thr107, T6: Ser147-Gly150);
(iii) α-helices (A1: Phe 25-Val 29, A2: Pro 30-The 41, A3: Gly 135-Gly 146). A loop
between B6 and B7 (Lys118-His126) and four β-strands (B3-B6) constitute a pocket for
CsA binding and PPIase activity (Pflugl et al., 1993; Hur and Bruice, 2002) (Fig 1.9).
A B Fig 1.9 Structure of the main domain and binding site of Cyp (Theriault et al., 1993; Hur and Bruice, 2002). A: Loops L1 to L4 form a channel for substrate binding; B: Residues for PPIase activity and CsA contact indicated in orange. Numbering of beta sheets or alpha helices seen in both.
Thirteen residues are involved in CsA contact by human CypA (hCypA): Arg55, Phe60,
Met61, Gln63, Gly72, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122 and
His126 (Pflugl et al., 1993); of these, Trp121 is essential (Bossard et al., 1991). Nine of
these residues (Arg55, Phe60, Gln63, Ala101, Asn102, Gln111, Phe113, Leu122,
His126) plus Ile57 and Arg148 are involved in PPIase activity (Pflugl et al., 1993;
Rascher et al., 1998), Arg55, Phe60 and His126 being essential for catalysis (Zydowsky
15
et al., 1992). Binding of calcineurin (CN) to the CsA-CypA complex increases the
stability and avoid proteolytic cleavage of the complex (Hornbogen et al., 1992), and
Arg69, Lys125 and Arg148 are responsible for CN contact (Theriault et al., 1993;
Rascher et al., 1998). Cyps from diverse organisms show a high degree of conservation
in the PPIase and CsA contact areas including Arg55, Phe60, Trp121, and His126
(Theriault et al., 1993; Galat, 1999). The active site is formed by four β-sheets and
residues on the enzyme surface, the loops forming a narrow channel for substrate
binding (Hur and Bruice, 2002). Catalysis may involve stabilisation of transaction state
at the binding site by hydrophobic interactions and stable H-bonding (Hamelberg and
Mecammon, 2009).
1.3.5 Composition of the human cyclophilin family
The human Cyp family is comprised of 19 members, falling into 10 classes (Galat,
2004) (representatives shown Fig 1.10; see legend for Genbank accession numbers).
Class A (hCyP18-a, b, c, ci, d) contains only the main domain, and two of these
(hCyP18-a, b) are possibly cytoplasmic. Class B contains N-terminal precursor
sequences and the main domain and has three members (hCyP-22b/p, hCyP-22c/p,
hCyP22d/p) predicted to be in the ER, membrane and mitochondria, respectively, with
hCyP-22b/p (Galat, 2004) (earlier called hCyPB, Price et al., 1991) showing the C-
terminal ER retention/retrieval signal (Freedman et al., 2002; Boldbaatar et al., 2008).
Class C (hCyP-19) has only the main domain while Class D members (hCyP-33, hCyP-
35) have an additional N-terminal RNA-binding domain, but unclear location (Galat,
2004; Wang et al., 2008). The class F member hCYP-40 has a C-terminal TPR
(tetratricopeptide repeat) domain, and the Class I member (hCyP-57) has a C-terminal
RNA-binding domain, in addition to their main domains. The two class G members
(hCyP-58, hCyP-58i) contain additional unknown domains at both termini. The class J
member hCyP73 has WD40 motifs, the two class H members (hCyP-88, hCyP-157)
exhibit zinc finger domains, and hCyP-157 is predicted to be in the nuclear membrane,
as also hCyP-358 of class E (Galat, 2004).
16
Fig 1.10 Schematic representation of cyclophilin members in human (Galat, 2004). Frames: main domains; Green ovals: precursor sequences; red bars in class D, E and I: RNA-binding domain; yellow: WD40 motifs; violet: zinc finger domain; orange: TPR domain; salmon: other domains. Only representatives of some classes are shown. (Class A: hCyP18a, b, c, ci, d; Genbank accession numbers NP_066953, NP_839944, NP_115861, NP_570981, NP_057143); class B: hCyP-22b/p, hCyP22d/p, hCyP-22c/p (NP_000933, NP_000934, NP_005720); class C: hCyP-19 (NP_006338); class D: hCyP-33, hCyP-35 (NP_006103; NP_775943); class E: hCyP-358 (NP_006258); class F: hCYP-40 (NP_005029); class G: hCyP-58, hCyP-58i (NP_680480, NP_680481); class H: hCyP-88, hCyP-157 (NP_004783, NP_005376); class I: hCyP-57 (NP_624311); class J: hCyP73 (NP_056157).
1.3.6 Diverse functions of Cyp in humans and other organisms (excluding plants)
As mentioned earlier, the main function of Cyps is to catalyse the cis/trans
isomerisation of Xaa-Pro peptide bonds in protein folding in vertebrates (Fischer et al.,
1984), a role also demonstrated in yeast and bacteria (Schonbrunner et al., 1991).
However, several additional functions have been reported for Cyps in vertebrates and
other organisms; these are summarised in Table 1.1. Briefly, the human hCyP18-a
(hCypA) may have a critical role in the maturation of homo-oligomeric receptors, as a
prolyl isomerase or a chaperone (Wang and Heitman, 2005). The membrane-located
hCyP-22c/p binds to unfolded protein substrates and acts as a chaperone to take proteins
for plasma membrane construction (Price et al., 1994; Meunier et al., 2002). The ER-
localised hCyP-22b/p, with prolactin complex, acts as a transcriptional inducer
(Rycyzyn and Clevenger, 2002) and also cooperates with other chaperones (PDI, Bip,
Grp94) to play crucial roles in protecting cells against ER stress (Kim et al., 2008). The
human 150 kDa Cyp (NK-tumor recognition molecule, NK-TR) containing several
domains and three regions containing Ser/Arg rich repeats have been identified in this
Cyp (Anderson et al., 1993). The human nuclear-localised SRcyp contains
serine/arginine dipeptide repeats in the C-terminal and is responsible for cell cycle
17
regulation (Dubourg et al., 2004). The anti-chaperone role of mouse CypB with PDI
and Hsp47 can export proteins from ER (Smith et al., 1995). The mitochondrial CypD
(homologue of hCyP22d/p) in rat glioma C6 cells functions in protection against
apoptosis (Lin and Lechleiter, 2002). The mammalian CyP40 containing TRP interacts
with heat shock protein (hsp90) (Ratajczak et al., 1993) and shows regulation of gene
transcription, transformation and differentiation (Leverson and Ness, 1998), chaperone
functions (Mok et al., 2006), and signaling of aryl hydrocarbon receptor (AhR) (Luu et
al., 2008).
The cytosolic CypA (TbCypA) from African trypanosomes may play role in the
interaction with infected species (Pelle et al., 2002) and has a 10 aa N-terminal
extension and a 3 aa insertion (KAG) in the centre. The Plasmodium falciparum PfCyP
contains an N-terminal signal peptide and two N-glycosylation sites (N120 and N137)
(Hirtzlin et al., 1995) corresponding to N71 and N108 of hCypA. The ER-localised
cyclophilin B in the fungus Aspergillus nidulans has a potential role in ER response
(Joseph et al., 1999). Expression of yeast CYP1 in the cytosol and CYP2 in the
secretory pathway under heat shock shows their roles in stress response (Sykes et al.,
1993) and in modulating the activity of histone-deacetylase complexes in transcriptional
events (Wang and Heitman, 2005). The yeast nuclear-localised Cyp (Rct1) functions in
pre-mRNA processing through maintaining correct phosphorylation of its own C-
terminal domain (CTD) (Gullerova et al., 2007). The plant Cyps are discussed
separately below.
18
Table 1.1 Key properties and functions of cyclophilins
Name location*/
Size (KDa) Species/cell
type Structure and /or Functions References
Cyp Cytosol/ 15 bovine Xaa-Proline isomerase, Trp residue essential for CsA binding
Handschumacher et al., 1984
Cyp Cytosol/ 18
bovine Regulation of T lymphocyte activation and proliferation
Harding et al., 1986
CyPA Cytosol/ na Human T cells Signal reception Brazin et al., 2002
CypB ER/ na bovine Cyp B increased the chaperone activity of PDI in vitro
Horibe et al., 2002
CyPB ER, PM / 21 human Chaperone to certain proteins destined for PM or export
Price et al.,1994; Price et al., 1991
CypB ER/ na human T47D With prolactin, acts as transcriptional
inducer for gene expression Rycyzyn and Clevenger, 2002
NK-TR Nucleus/ 150 human Identified in natural killer cells Anderson et al., 1993
SRcyp Nucleus/ na human Contains S/R repeat at C-terminus with function for cell cycle regulation
Dubourg et al., 2004
CyPB ER/ na mouse 3T6 cells
Anti-chaperone, export procollagen from ER, cooperating with Hsp47 and PDI
Smith et al., 1995
CypB ER/ na mouse ER chaperone forms an ER network binding to unfolded protein substrates; protecting cells against ER stress via its PPIase activity.
Meunier et al., 2002; Kim et al., 2008
CypD MT/ na mouse Protection against apoptosis Baines et al., 2005; Lin et al., 2002
CyP40 Nucleus/ 40 mammalian cells
Regulation of gene; role in AhR signaling; 186–215aa essential for forming AhR/Arnt/DRE complex; TPR domain has chaperone function
Leverson et al., 1998; Mok et al. 2006; Luu et al., 2008
CypB ER/na insect cells Stimulates NS5B-catalysed RNA synthesis
Heck et al., 2009
CypG ER/ 23 horseshoe crab
Chaperone to take stored proteins in exocytotic granules
Takaki et al., 1997
CRIP Nucleus /na
Paramecium Cyclophilin-RNA interacting proteins, cell morphogenesis, cortical organization and nuclear reorganization
Krzywicka et al., 2001
TbCypA Cytosol/ 18.7 Trypanosoma Involved in interactions with infected species
Pelle et al., 2002
CypB ER/ na S. cerevisiae Has HDEL signal for ER-localisation Frigerio and Pelham , 1993
CYP1, CYP2
Cytosol / na, secretory / na
S. cerevisiae Function in stress conditions Sykes et al., 1993
Rct1
Nucleus / na
S. pombe RRM-containing cyclophilin, pre-mRNA processing
Gullerova et al., 2007
*ER: endoplasmic reticulum, MT: mitochondria; PM: plasma membrane.
1.4 Properties and functions of plant cyclophilins
1.4.1 Cyclophilin family in Arabidopsis: a summary
The 29 Cyp isoforms identified from Arabidopsis genome range from 17.5-95 kDa and
are predicted to be in the cytosol (13), chloroplast (6), ER (5), mitochondria (2) and
nucleus (3) (He et al., 2004; Romano et al., 2004a). Their key features are summarised
below due to relevance for comparative purposes (Fig 1.11; Table 1.2) (nomenclature
according to Romano et al., 2004a). Nine cytosolic Cyps (AtCYP18-1, AtCYP18-2,
AtCYP18-3, AtCYP18-4, AtCYP19-1, AtCYP19-2, AtCYP19-3, AtCYP22, AtCYP26-
1) contain only the main domain, 4 are multidomain, and genes of six (AtCYP18-3,
AtCYP18-4, AtCYP19-1, AtCYP19-2, AtCYP19-3, AtCYP26-1) are intronless (Chou
19
and Gasser, 1997; He et al., 2004). AtCYP40 contains the main domain (residues 1-
174) at N-terminus and a TPR (264-331) (He et al., 2004) and is required for vegetative
phase change (Berardini et al., 2001) through regulation of miR156-regulated members
(Smith et al., 2009). AtCYP57 contains a C-terminal Arg/Lys rich domain (200-504)
and AtCYP65 contains a U-box (38-97) at N-terminus and main domain (343-498). U-
box is a modified RING finger domain and may required for protection from protein
degradation (reviewed in He et al., 2004). AtCYP65 acts as a prolyl isomerase and a
chaperone for resolving protein aggregates (AtPUB49; Wiborg et al., 2008). AtCYP71
contains the main domain (475-631) and WD repeats (66-240) at N-terminus and acts as
a histone remodeling factor involved in gene silencing (Li et al., 2007). WD-40 repeat
contains a central Trp-Asp presenting as tandem repeats and its function is for protein-
protein interaction (reviewed in He et al., 2004).
Fig 1.11 Structures of putative representative cyclophilins in Arabidopsis. Based on He et al. (2004). Dark grey boxes: Cyp domains; ER: ER targeting signal, L-Z: leucine zipper, RRM: RNA recognition motif, TPR: tetratricopeptide repeats, Z-F: zinc finger, C/T: chloroplast/thylakoid lumen targeting signal, MT: mitochondrial targeting signal; U-box, WD repeats.
The five predicted ER-Cyps (AtCYP19-4, AtCYP20-1, AtCYP21-1, AtCYP21-2,
AtCYP23) all contain N-terminal ER-targeting signals (residues 1-23, 1-23, 1-27, 1-31,
1-22) and C-terminal main domains (36-201, 37-204, 43-224, 60-228, 31-196) (He et
al., 2004). AtCYP19-4 localises in the ER (AtCYP5; Saito et al., 1999) and regulates
the protein GNOM required for coordination of cell polarity along the embryo axis
(Grebe et al., 2000). AtCYP20-1 functions in regulating protein phosphatase 2A
20
(PP2A) for auxin transport and growth response (Jackson and Soll, 1999) and AtCYP23
shows a C-terminal extension (197-226).
The six chloroplastic isoforms (AtCYP20-2, AtCYP20-3, AtCYP26-2, AtCYP28,
AtCYP37, AtCYP38) contain chloroplast/thylakoid (CT) lumen targeting sequence (1-
76, 1-79, 1-73, 1-33, 1-114, 1-92) and main domain (85-259, 95-260, 93-317, 77-290,
281-422, 260-437) (He et al., 2004; Romano et al., 2004a). AtCYP20-2 plays an
important role in catalysing the correct folding and integration of proteins in the
thylakoid membrane (TLP20, Edvardsson et al., 2003; Romano et al., 2004b) and shows
PPIase activity (Shapiguzov et al., 2006). AtCYP20-3 also shows this activity and
localises in the stroma (ROC4; Lippuner et al., 1994). Its disulfides (Cys54-Cys171,
Cys129-Cys176) are important for its isomerisation and disulfide reduction activities
(Laxa et al., 2007) and it links the photosynthetic electron transport and redox
regulation to folding of SAT1 (serine acetyltransferase, a rate-limiting enzyme in Cys
biosynthesis) (Dominguez-Solis et al., 2008). Roles of AtCYP20-3 include repair of
photodamaged photosystem II (PSII) (Cai et al., 2008). AtCYP37 has a C-terminal
extension (423-461) while AtCYP38 has a leucine zipper (111-170) (He et al., 2004;
Romano et al., 2004a) and a critical role in the assembly and maintenance of PSII
supercomplexes (Fu et al., 2007; Sirpio et al., 2008). The mitochondrial Cyps
(AtCYP21-3, AtCYP21-4) have N-terminal targeting signals (both 1-47) and main
domain (77-230, 80-236, respectively) (He et al., 2004; Romano et al., 2004a).
The three nuclear isoforms (AtCYP59, AtCYP63, AtCYP95; main domains 1-161, 1-
180; 1-174) (He et al., 2004; Romano et al., 2004a) have had their location confirmed
by GFP fluorescence (Lorkovi et al., 2004; Gullerova et al., 2006). AtCYP59 contains
an RNA recognition motif (RRM) (243-321), a zinc finger motif (342-355) and a C-
terminal Arg/Lys rich domain (397-506); the latter contains a RS/RD(E) rich motif
essential for nuclear localisation, and is involved in connecting transcription and pre-
mRNA processing (Gullerova et al., 2006). AtCYP63 and AtCYP95 contain an
Arg/Lys domain (228-570; 348-837) and play a role in mRNA splicing, possibly by
regulating the phosphorylation/ dephosphorylation of signal recognition proteins and
other spliceosome components (Lorkovi et al., 2004).
21
Table 1.2 Properties of cyclophilin family in Arabidopsis and their orthologues
Name Other names
Functions (other properties) References
AtCYP18-1 Regulation of K+ channels in guard cell; enhances multiple abiotic stress tolerance; PPIase activity of their orthologue in tomato is confirmed and its function is for auxin regulation of plant growth, development and environmental responses; Acts during development; participation in response to salt stressful conditions.
Luan et al., 1993; Kumari et al., 2009; Oh et al., 2006; Kielbowicz-Matuk et al., 2007; Chen et al., 2007
AtCYP18-2 AtCYP18-3 ROCI
AtCYP18-4 ROC5, AtCYP1 AtCYP19-1 ROC3
AtCYP19-2 ROC6
AtCYP19-3 ROC2 AtCYP22 n/a n/a
AtCYP26-1 n/a n/a
AtCYP40 Regulates microRNA activity for vegetative phase change
Berardini et al., 2001; Smith et al., 2009
AtCYP57 n/a n/a
AtCYP65 AtPUB49 (65kDa)
AtPUB49 with U-box functions both as a PPIase and a chaperone, catalysing isomerisation of peptidyl-prolyl bonds and resolving protein aggregates
Wiborg et al., 2008
AtCYP71 Serves as a highly conserved histone remodeling factor involved in chromatin-based gene silencing
Li et al., 2007
AtCYP19-4 Cyp5 (19 kDa)
regulates GNOM coordinating cell polarity Grebe et al., 2000
AtCYP20-1 ROC7 (22 kDa)
Regulating PP2A activity for auxin transport and growth response pathways
Jackson and Soll, 1999
AtCYP21-1 n/a n/a AtCYP21-2 n/a n/a AtCYP23 n/a n/a AtCYP20-2 TLP20 (20kDa)
Important role in catalysing correct folding and integration of proteins in and around the thylakoid membrane system; Catalysis of chloroplast protein folding in thylakoid lumen; PPIase activity in thylakoid lumen restricted to only AtFKBP13 and AtCYP20-2
Romano et al., 2004b; Edvardsson et al., 2003; Shapiguzov et al., 2006
AtCYP20-3 ROC4 (20kDa)
Active PPIase localised in the stroma of chloroplast; ROC4 functions in the repair of photodamaged PSII (chloroplast stroma); Disulfide bonds are very important for isomerisation and disulfide-reduction activities CYP20-3 links photosynthetic electron transport and redox regulation to the folding of SAT1.
Lippuner et al., 1994; Cai et al., 2008; Laxa et al., 2007; Dominguez-Solis et al., 2008
AtCYP26-2 n/a n/a
AtCYP28 n/a n/a AtCYP37 n/a n/a
AtCYP38 Plays a critical role in assembly and maintenance of photosystem II supercomplexes during PSII assembly
Fu et al., 2007; Sirpio et al. 2008
AtCYP59 Activities connecting transcription and pre-mRNA
processing Gullerova et al., 2006
AtCYP63; CypRS64 (64kDa)
Nuclear location confirmed by GFP fluorescence; Function for pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components
Lorkovi et al., 2004
AtCYP95 CypRS92
AtCYP21-3 n/a n/a
AtCYP21-4 Involved in the resistance mechanism of one of the most important diseases of wheat, yellow rust
Bozkurt et al., 2008
22
1.4.2 Properties and functions of Cyps in other plants
CypA cDNAs from the tomato were initially shown to be expressed in all parts of the
plant and the protein sequence showed 83% conservation to Cyp from vertebrates
(Gasser et al., 1990). The spinach 40kDa Cyp (TLP40; homologue of AtCYP38) in the
thylakoid lumen plays a dual role in photosynthesis involving biogenesis and intra-
organelle signaling (Fulgosi et al., 1998). The 18 and 20 kDa Cyps in Vicia faba are
responsible for regulation of K+ channels in guard cell or under stress conditions (Luan
et al., 1993; Godoy et al., 2000). The 20 kDa plays a specific role in chloroplast
through a unique mode of regulation (Luan et al., 1994a; 1994b). The expression of
gene coding 18 kDa StCyP (AY972080) in potato (Solanum tuberosum) is stimulated
by wounding, fungal infection and heat-shock, showing an involvement in stress
response (Godoy et al., 2000) and also plays an important role in stress responses
induced by salicylic acid and pathogen (Phytophthora infestans) elicitor and infection
(Dubery, 2007). The tomato 17.9 kDa LeCYP1 shows a role in auxin regulation of
plant growth, development, and environmental effects (Oh et al., 2006). A Cyp
(ThCYP1) in salt cress (Thellungiella halophila) shares 90% identity with AtCYP19-1
and 81% identity with LeCYP1 on amino acid level, and participates in response to salt
stressful conditions (Chen et al., 2007). SsCyp (17.9 kDa) of eggplant (Solanum
sogarandinum) show high homology to AtCYP19-1, AtCYP18-3 and AtCYP18-4, has
89% identity with StCyp (AAD22975) of S. tuberosum (Godoy et al., 2000) and plays a
role in plant development (Kielbowicz-Matuk et al., 2007). A 1kb cNDA encoding
GjCyp-1 from a red alga (Griffithsia japonica) was shown to have a role in influencing
plant growth and development in a tobacco transgenic line (Cho and Kim, 2008).
Cytosolic (17.5 kDa, cytosolic PPI) and microsomal (17.7 kDa, microsomal PPI) Cyps
have been isolated from maize and their PPIase activity is inhibited by CypA. The high
similarity (~60% identity) of the N-terminal sequence
(KKDLTEVTHKVYFDV/IEIDGKPAGRIVMGLF) from the purified maize
microsomal Cyp with that of mature animal s-cyclophilin (cyclophilin B) strongly
suggests it is a microsomal cyclophilin B (Sheldon and Venis, 1996). 40 kDa Cyp
(TLP40) contains N-terminal leucine zipper for interaction with other leucine zipper
proteins. It is located in the lumen of the thylakoids and plays a major role in
photosynthesis involving both biogenesis and intraorganelle signalling (Fulgosi et al.,
1998). The drought stress-induced enhancement of Cyp (20 kDa, SorgCyp20)
23
expression on protein level in both leaves and seeds differs between the tolerant and
susceptible cultivars of sorghum (Sorghum bicolor L Moench) (Sharma and Singh,
2003). Further, the stress-induced enhancement of PPIase activity of Cyps depends
upon the tissues, types of stress treatment, and cultivars, e.g. draught-induced PPIase
activity increases in the leaves of the tolerant cultivars and decreases in susceptible
cultivar (Sharma et al., 2004; Sharma and Singh, 2006).
Limited information existed about the Cyp family in rice at the start of this project.
Only two members (OsCyp1 and OsCyp2) had been reported. Cyp1 expresses in leaves
and/or root, and Cyp2 expresses in both leaves and roots. Genomic Cyp2 is intronless
(Buchholz et al., 1994). Expression of the gene encoding the 172 aa cytosolic OsCyp2
(EF576508) is stimulated by salinity stress (Kumari et al., 2009). Purified OsCyp2
protein exhibits CsA sensitive PPIase activity and expression of OsCyp2 in E. coli
shows it functions in multiple abiotic stress tolerance including salinity, high
temperature, osmotic and oxidative stresses (Kumari et al., 2009).
Both the transcripts of prolamin storage proteins and the transcription of PPIase are
detected 14 days post-anthesis in developing wheat endosperm, which implies PPIase
may play important roles in storage protein formation even if the particular PPIase
involved in these roles have not been identified (Grimwade et al., 1996). A Cyp-like
protein in wheat is involved in mechanism of race-specific resistance to races of yellow
rust (Puccinia striiformis f. sp. tritici ) and is homologous to AtCYP21-4
(NP_187319.1, Romano et al., 2004a) (Romano et al., 2004a; Bozkurt et al., 2008). M.
Bhave’s group previously characterised genes encoding cytoplasmic CypAs in wheat,
which were found to be located on chromosome 6AS, 6BS and 6DS and intronless
(Johnson et al., 2004b). These may be involved in the translocation of storage proteins
into ER. Their cytoplasmic location makes a direct role in protein folding inside the ER
unlikely. However, little other information exists on ER localised or other Cyp
isoforms in wheat.
24
Table 1.3 Key properties of cyclophilins in other plants
Name Cellular
location/ Size
Species Functions and other properties References
Cyp Na/na P. vulgaris . cv. Saxa
Abiotic stresses (heat, cold) Marivet et al., 1994
Cyclophilin Chloroplast/na pea Folding membrane proteins Breiman et al., 1992
CyPs Na/18kDa; 21kDa
V. faba Regulation K+ channels in guard cell
Luan et al., 1993
pCyP B/C Chloroplast/ 21kDa
V. faba Role in chloroplast function Luan et al., 1994a
pCyPB Chloroplast/ 21kDa
V. faba Expression induced by heat shock
Luan et al., 1994b
TLP40 Thylakoid lumen/40kDa
spinach
Dual role in photosynthesis : biogenesis and intra-organelle signaling
Fulgosi et al., 1998
StCyP Na/18kDa potato Role for cyclophilins in the plant response to environmental stresses
Godoy et al., 2000
Cyclophilin (CyP)
Na/18 kDa potato CyPs play an important role in plant stress responses induced by pathogen
Dubery 2007
CYP, LeCYP1
Na/17.9kDa tomato Auxin regulation of plant growth, development, environmental responses
Oh et al., 2006
SsCyPs Cytosol/ 17.9 kDa
eggplant Acts during plant development
Kielbowicz-Matuk et al., 2007
GjCyp-1
Na/na Transgenic tobacco
Influence growth and development
Cho and Kim 2008
ThCYP1 Nucleus/173 aa
salt cress response to salt stressful condition
Chen et al., 2007
cyclophilins Cytosol/17.5 kDa; ER/17.7 kDa
maize PPIase activity; microsomal Cyp Sheldon and Venis, 1996
OsCyp2 172 aa rice Enhances multiple abiotic stress tolerance
Kumari et al., 2009
Cyclophilin like protein
Na/na wheat Involved in mechanism of resistance to wheat yellow rust
Bozkurt et al., 2008
1.5 Protein disulfide isomerase (PDI)
1.5.1 Discovery of PDI
The enzyme protein disulfide isomerase (PDI; EC 5.3.4.1) was initially found to
catalyse the inactive and reduced ribonuclease A (RNase A) to its active form in bovine
liver (Goldberger et al., 1964); it was then reported from many other organisms
including yeast (LaMantia et al., 1991) and plants (Coughlan et al., 1996). PDI is now
categorised as belonging to a superfamily, consisting of five members in yeast
(Norgaard et al., 2001), 19 in humans (Appenzeller-Herzog and Ellgaard, 2008a) and
19-22 in plants (Houston et al., 2005).
25
1.5.2 PDI as a member of the thioredoxin superfamily
Thioredoxin (TRX) is a small ubiquitous protein which contains an active site with a
functional motif ‘WCXXC’ (Holmgren, 1985). Proteins catalysing redox reactions,
containing one or more copies of the highly conserved TRX active site motif WCXXC
and showing sequence similarity to TRX are included in the TRX superfamily (Ferrari
and Soling, 1999; Gruber et al., 2006). Based on the numbers of secondary subunits
and variations in motifs at the catalytic site, the TRX superfamily is divided into four
families (Fig 1.12) (Martin, 1995; Jacquot et al., 2002; Houston et al., 2005):
• Thioredoxins (TRXs): approximately 12 kDa (~110 aa) proteins with a very
conserved active site motif with the sequence WC(G/P)PC (Jacquot et al., 2002)
• Protein disulphide isomerase-like proteins (PDILs), which include three groups: (i)
protein disulphide isomerase (PDI); (ii) the quiescin-sulfhydryl oxidase-like
(QSOXL) group; (iii) the adenosine 5’-phosphosulfate reductase-like (APRL) group
(reviewed in Houston et al., 2005). PDIs contain the active site motif WCGHC
(Kemmink et al., 1997). QSOXLs contain a TRX motif WCXXC at N-terminus and
a Erv1-like domain at C-terminus for sulfhydryl oxidase activity, confirmed in
vertebrates (Lee et al., 2000; Thorpe et al., 2002). The APRLs contain N-terminal
chloroplast targeting peptides, an adenylyl sulfate reductase (APR) activity core and
a C-terminal TRX motif WCXXC (Prior et al., 1999)
• Glutaredoxins (GRXs) with the TRX motif YCP(Y/F)C (Jacquot et al., 2002)
• Peroxi-redoxins (PRXs) with a single catalytic Cys (Jacquot et al., 2002).
Of these, the PDILs are of relevance to the project, and are detailed below.
26
TRX Superfamily
PDI (WCGHC)
TRX (WCG/PPC)
GRX (YCPY/FC)
PRX (single C)
Class 1 (2 a-like
domains at N-and C-
terminus)
Class 2 (2 a-like
domain in middle)
Class 3 (3 a-like domains, 2 at N- and 1 at C-terminus
Class 4 (3 a-like domain at C-terminus)
Class 5 (1 a-like domain)
PDILs
QSOXL (WCXXC; Erv1-like)
APRL (WCXXC;
APR)
Vertebrate (N), plant?
Vertebrate (Y)
Vertebrate (Y), Plant (Y)
Fig 1.12 Classification of the TRX superfamily (Based on Martin, 1995; Jacquot et al., 2002; Houston et al., 2005).
1.5.3 Structure of PDI
The PDI is approximately 500 amino acids or 60 kDa in size. Molecular masses of
purified PDIs differ slightly between different organisms, e.g., 57 kDa (bovine)
(Carmicheal et al., 1997), 60 kDa (yeast) (Mizunaga et al., 1990), 55 kDa (castor bean)
(Coughlan et al., 1996), 60 kDa (wheat) (Shimoni et al., 1995b) or 63 kDa (soybean)
(Kainuma et al., 1995). Using proteolysis of native PDI and characterisation of peptide
fragments, combined with bioinformatics approaches, the currently accepted structure of
PDI has four domains (a, b, b' and a'), x-link, and a C-terminal acidic extension
(Freedman et al., 2002; Wilkinson and Gilbert, 2004) (Fig 1.13a). The boundary
numbers of domains are based on sequences expressed from human PDI cDNA, with
first expressed residue being Asp1 (Darby and Creighton, 1995; Darby et al., 1998).
The a and a’ domains contain active sites WCGHC, which mediates the activity of PDI.
Domains b and b’ also adopt a TRX fold, but do not share high sequence similarity with
each other or with a or a’. The C-terminal contains an ER-retention signal ‘KDEL’ in
human (Denecke et al., 1992; Freedman et al., 2002) and ‘HDEL’ in yeast (Frigerio et
al., 1993; Grube et al., 2006). The structures of a and b domains have been determined
by nuclear magnetic resonance techniques (Kemmink et al., 1997) and are similar, with
five-stranded parallel or anti-parallel β-sheets in the centre, surrounded by four α-
helices (Fig 1.13b). Domains a and a’ contain an extra section (grey extension shown
27
by arrow in Fig. 1.13b) which has catalytic activity (Vuori et al., 1992). However, their
isomerase activities depend upon presence of b, and b’. All single TRX domain
members exhibit the characteristic α/β fold (reviewed in Freedman et al., 2002).
WCGHC
a b b’x
a’ c
WCGHC
7-117 119-216 218-332 352-462
KDEL
491
(a)
(b)
Fig 1.13 Domains organisation of human PDI (Freedman et al., 2002). (a) Structure of human PDI, consisting of four domains, one linker, and C-terminal extension, organized as a-b-b’-x-a’-c. (b) Secondary structures of PDI domains: A: a-domain; grey extension responsible for catalytic activities indicated by the arrow; B: b-domain.
1.5.4 The primary biochemical activity of PDI
As mentioned above, PDI was first found to catalyse the inactive and reduced RNase A
to its active form (Goldberger et al., 1964). PDI primarily catalyses the refolding and
unfolding of nascent proteins by disulfide bond breakage, formation and rearrangement
in eukaryotes (Creighton et al., 1980). A 52 kDa microsomal PDI binds to different
secretory proteins after their translocation into ER, and also binds to proteins with no
Cys (Klappa et al., 1995). It has one active site each at the N- and C- terminus, both
containing the catalytic motif WCGHC. The two Cys can either form an intra-
molecular disulfide (PDI in oxidised state); or exist in dithiol form (PDI in reduced
state) (Fig 1.14). The oxidase activity of PDI is responsible for disulfide formation in
the early stages of protein folding, while the reductase activity can accelerate the
isomerisation of incorrect disulfides (Wilkinson and Gilbert, 2004). PDI with the motif
WCGHS cannot catalyse reduction or oxidation, but can catalyse disulfide isomerisation
(Woycechowsky et al., 2000; Serrato et al., 2008). The active sites cannot completely
catalyse substrate oxidation or reduction in vivo, as shown by the distribution of human
28
PDI in HEK-293 cells (50% fully reduced, 18% a-oxidised/a’-reduced, 15% a-
reduced/a’-oxidised, 16% fully oxidised) (Appenzeller-Herzog and Ellgaard, 2008b).
SH SHPDI
S S
PDI
SH SHS S
sulfhydryl disulfide
S S
S S
S
S
S
S
PDI
SH SH
PDI
Disulfide formation
SH SH
Disulfide isomerization
Oxidized
PDI
Reduced
PDI
Fig 1.14 Disulfide formation and isomerisation accelerated by PDI (Wilkinson and Gilbert,
2004). The process of disulfide formation of native bonds is shown in Fig 1.15. PDI is
oxidised by ER oxidoreductin proteins before performing its disulfide formation
function (Woycechowsky et al., 2000). Such proteins include Ero1-Lα and Ero1-Lβb in
mammalian cells, AERO1 and AERO2 in Arabidopsis (Dixon et al., 2003), and Ero1p
or Erv2p in yeast (reviewed in Gruber et al. 2006). A domain-swap study shows that the
b'xa' fragment of hPDI provides the binding region for Ero1-Lα with Cys pair (Cys94-
Cys99) (or Cys100-Cys105 in Ero1p) (Wang et al., 2009) (Fig 1.16). Reduced PDI,
generated by oxidants, is generally an additional requirement for efficient oxidative
folding (Lyles and Gilbert, 1991; Rancy and Thorpe, 2008).
Oxidized state
Reduced state
Fig 1.15 Mechanism of disulfide bond formation in native proteins (Woycechowsky et al.,
2000).
29
Fig 1.16 Schematic model illustrating the interaction between Ero1-L and hPDI (Wang et al., 2009).
The role of disulfide bond formation in nascent proteins is fulfilled by a combination of
active and inactive TRX sites. The thiol-disulfides exchange could be catalysed by PDI
homologues containing either a’- or a’-domain, but disulfide isomerisation requires
these catalytic domains plus the b and b’-domain in hPDI (Freedman et al., 2002). The
b domain appears to provide the main peptide binding site, e.g., holding the substrate
protein in an unfolded conformation for the catalytic sites to perform the thiol-disulfide
exchange (Freedman et al., 2002), and there is no experimental evidence for a role for b
in substrate binding (Appella and Anderson, 2007; Hatahet and Ruddock, 2007). The
linker region x inhibits the substrate binding of b’-domain by interaction of the
hydrophobic residues of x with b’ (Nguyen et al., 2008).
1.5.5 Composition and function of human PDIL family members
The PDIL families in many organisms have been found to exhibit a great diversity of
structures and functions. The human PDI-like (PDIL) family is comprised of 18
members (17 PDI, one QSOX), all containing at least one TRX-like domain (reviewed
in Ferrari and Soling, 1999; Thorpe et al. 2002; Hatahet and Ruddock, 2007) (Fig 1.17;
see legend for Genbank accession numbers). Some key features are described below to
illustrate the structural and functional complexity and variability, which are of relevance
to the present work.
Seven members (PDI, ERp57, ERp72, PDIr, ERdj5, PDIp, PDILT) contain four or more
TRX-like domains, of which four members (PDI, ERp57, PDIp, PDILT) have all four
main domains (a, b, a’, b’). The putative human PDI (hPDI) (508 amino acids) includes
domains a (residues 23-130), b (135-230), b’ (235-344), and a’ (368-471) (boundary
numbers counted from the first residue Asp1; Darby and Creighton, 1995; Darby et al.,
30
1998). The putative ERp57 (505 amino acids) also contains these domains (25-129,
134-237, 241-354, 377-481). ERp72 (645 aa) contains five: ao (61-166), a (175-280), b
(286-390), b’ (395-503), and a’ (625-632), where all three a-like domains have the
characteristic WCGHC. PDIr (519 aa) presents the b-domain (28-136) at N-terminus
and three a-like domains (ao: 150-257, a: 275-380, a’: 396-502) (Ferrari and Soling
1999) with variant motifs (CSMC, CGHC, CPHC) (reviewed in Ellgaard and Ruddock,
2005; Hatahet and Ruddock 2007). The ER-localised ERdj5 has four a-type domains,
also with variant motifs (CSHC, CPPC, CHPC, CGPC). In terms of biochemical
activities, PDI and ERp72 demonstrate all activities (oxidase, reductase, isomerase,
chaperone) (Wilson et al., 1998; Forster et al., 2006); PDIp and PDIr exhibit three
(oxidase/reductase/chaperone and reductase/isomerase/chaperone, respectively)
(Hayano and Kikuchi, 1995; Klappa et al., 2001); ERp57 presents two (oxidase,
reductase) (Obeid, 2008); ERdj5 only the reductase activity (Ushioda et al., 2008); and
the activity of PDILT is unknown as yet (van Lith et al., 2005) (reviewed in Kim et al.,
2009).
Fig 1.17 Domains in human PDI members (Hatahet and Ruddock, 2007). Yellow box indicates a-domain and a’-domain, red box indicates b-domain, and green box indicates b’-domain, grey stick indicates connection between domains, and the rectangular box in some indicates x link. (Genbank accession numbers; PDI: P07237; ERp57: P30101; PDIp: Q13087; ERp72: P13667; PDILT: Q8N807; ERp27, Q96DN0; PDIr: Q14554; ERp29: P30040; ERdj5: Q8IXB1; P5, Q15084; ERp18: O95881; ERp44: Q9BS26; ERp46: Q8NBS9; TMX: Q9H3N1; TMX2: Q9Y320; TMX3: Q96JJ7; TMX4: Q9H1E5).
31
ERp46 (432 aa) contains three a-like domains with the motif CGHC, and ERp44 (406
aa) has domains a (CRFS), b, and b’, but the activities of both proteins are unknown.
P5 (440 aa) presents a and a’ domains at N-terminus with oxidase, isomerase and
chaperone activities, and ERp27 (273 aa) has b and b’ but unknown activities. Only the
a-domain occurs in 5 members (Erp18, 172 aa; TMX, 280 aa; TMX2, 296 aa; TMX3,
454 aa; TMX4, 349 aa). Three members (Erp18, TMX, TMX3) exhibit oxidase and/or
isomerase activities. The mature ERp18 (146 aa, without the 26-residue signal peptide)
shows the C-terminal ER retention signal (EDEL and QDEL in human and mouse,
respectively) and is localised in ER lumen (Jeong et al., 2008). TMX (active site
CPAC) has reductase activity, and ERp29 (P30040, Ellgaard and Ruddock, 2005;
ERp28; X94910, Ferrari and Soling, 1999) contains b domain (35-146) with no
confirmed activity (reviewed in Ellgaard and Ruddock, 2005; Appenzeller-Herzogand
Ellgaard, 2008a; Kim et al. 2009).
The deduced sequence of human QSOX (HsQSOXL, HSU97276) contains an N-
terminal signal sequence, the a/a’ domain (WAV--ALA) of PDI with the motif
WCGHC, an ERV1 domain (GYV--CHN) including its three characteristic sections,
and a C-terminal variable part with no ER retention motif. The three sections in the
ERV1 domain are: (i) section 1 (GYV--QMA) with the active site (CXDC); (ii) section
2 (AVL--GAP) with consensus motif (HNXVNXRL) for interacting with the ADP
moiety of the FAD prosthetic group; (iii) section 3 (PPK--CHN) with the consensus
motif (ELCXXC) (Thorpe et al., 2002). There are no APRL members in human PDIL
superfamily; these only occur in plants and are explained below.
1.5.6 PDIL families in other organisms
The PDIL family comprises five members in yeast (Norgaard et al., 2001), of which
Pdi1p and Eug1p contain two a-like domains, the active site motifs of Eug1p being
CXXS which cannot form intramolecular disulfides. The other three (Mpd1p, Mpd2p,
and Eps1p) have one a-like domain with motifs CGHC, CQHC, or CPHC, respectively
(Norgaard et al., 2001) and Mpd1p may be functionally analogous to human ERp57
(Vitu et al., 2008). Transcriptomics analysis shows yeast PDI (Pdi1), with other
chaperones, has potential functions in the secretion machinery and ER stress regulation
32
in yeast (Gasser et al., 2007). PDI homologues are also reported in the fungus
(Neurospora crassa) (Tremmel et al., 2007).
The Chlamydomonas reinhardtii chloroplast PDI (RB60), by its phosphorylation and
redox activity, is involved in the light-regulated translation of psbA mRNA, which
encodes a core protein D1 of PSII through modulating RB47, a poly(A)-binding protein
( psbA) (Trebitsh et al., 2001; Alergand et al., 2006). The identification of RB60 by
proteomic approach in Chlamydomonas reinhardtii also indicates its oxidation activity
through the glutathionylation (Michelet et al., 2008). Eleven PDILs occur in the
protozoan parasite Entamoeba histolytica, falling into (i) one a domain; (ii) one a
domain with at least one b, and (iii) a and a’ with/without other domains (Ramos et al.,
2008). The plant PDIs are discussed separately below.
1.5.7 Functions of PDI in human and other organisms
Human PDI has been shown to conduct several functions through its main role in the
ER, i.e., catalysing both the oxidation and isomerisation of disulfide bonds of nascent
polypeptides. These functions include: catalysing the regeneration of ascorbic acid and
initiating the oxidation of intralumenal protein thiols to disulfides (Wells et al., 1990),
keeping the D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with no disulfide
bonds in its active state by suppressing its aggregation (Cai et al., 1994), and preventing
the aggregation of denatured rhodanese (Yao et al., 1997). PDI also performs
chaperone functions, e.g., refolding of denatured GAPDH (Cai et al., 1994 and
rhodanese with no disulfide bonds (Yao et al., 1997), and is also a subunit of prolyl 4-
hydroxylase and microsomal triglyceride transfer protein (reviewed in Noiva 1999).
PDI also functions in compartments other than ER lumen. In the cytoplasm,
endosomes, or cell surface, it catalyses the reduction of protein disulfides for
participation in the thyroxin-induced actin nucleation, or cell-to-cell interaction through
activation of integrins (Noiva 1999).
Three other hPDIs (ERp57, PDIp, ERp72) have roles as components of peptide-loading
complexes, peptide binding, and protein retention in ER. Other PDI roles include
immune evasion of tumor (P5), protein retention in ER, and regulation of IP3R1 activity
(ER44) (reviewed in Kim et al., 2009). ERp18 is responsible for apoptosis during ER
stress caused by accumulation of misfolded proteins (Jeong et al., 2008). ERdj5
33
functions in degradation of misfolded proteins through its reductase activity, cleaving
their disulfide bonds and accelerating ERAD (ER-associated degradation protein). It
performs this function in a complex with EDEM (ER degradation-enhancing a-
mannosidase-like protein) and ER-resident chaperone BiP (Ushioda et al., 2008) (see
section 1.5.5).
1.5.8 Additional functions of PDI relevant to cell biology and medicine
• PDI has been linked to transport of nitrous oxide to cell surface and cytosol and of the
estrogens into cells (Sliskovic et al., 2005; Fu et al., 2008).
• Quantitative proteomics shows its functions (with other chaperones) in apoptosis
against ER stress in human cell lines (Short et al., 2007).
• PDIp and PDI are critical for accumulation of misfolded proteins in neurodegenerative
diseases (Nakamura and Lipton, 2009).
• PDI activates of tissue factor (TF), a major initiator protein of blood coagulation
(Manukyan et al., 2008).
• PDI can be used for studies such as obesity, insulin, inflammation (Boden et al., 2008)
and tumor destruction (Fonseca et al., 2009).
• The anti-cancer drug Vincristine inhibits chaperone activity of hPDI and P5, showing
potential for new drug design (Horibe et al., 2008). Based on functions in ER-stress
and apoptosis, small-molecule PDI inhibitors may have potential for cancer treatment
(Lovat et al., 2008).
The diverse catalytic and chaperone activities of PDI in human and other organisms
(excluding plants) are summarised in Table 1.4. While a significant proportion of
information on PDILs comes from humans for historical and medical reasons, and
although some of the functions may not apply to other organisms, the range indicates
the capabilities of PDI far exceed a catalytic role alone. The universality of cellular
events or needs such as protein-protein complexes, ER stress, apoptosis, and signal
transduction, suggests the roles of PDI could be just as diverse in other organisms,
deserving further investigation. The plant PDIs are discussed below.
34
Table 1.4 Properties and functions of PDI in vertebrates and yeast
Species/tissues
Length (aa), domains
Function/Cellular location Reference
Rat/liver, n/a
Catalyse RNase A Goldberger et al., 1964
Recombined rat PDI n/a
Bind the hormones, estradiol, 3,3',5-triiodo-L-
thyronine (T3) Primm et al., 2001
Bovine/ Pancreas n/a
Refolding and unfolding protein by disulfide bond breakage, formation and rearrangement
Creighton et al., 1980
Bovine/ microsome n/a
Maintain catalysing form of triglyceride transfer protein (MTP) and prevent aggregation
Wetterau et al., 1991
Bovine/ liver n/a
Catalyse regeneration of ascorbic acid and initiate oxidation of intralumenal protein thiols to disulfides
Wells et al., 1990
Keep D-Glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) with no disulfide bonds in its active state by suppressing aggregation (Chaperone-like)
Cai et al., 1994
Prevent aggregation of guanidine-hydrochloride-denatured rhodanese (Chaperone-like)
Song and Wang, 1995
Human PDI (P07237) 508; a-b-b’-a’
Peptide-binding Klappa et al., 1998 Transport NO to cell surface and cytosol Sliskovic et al., 2005
Protein retrotranslocation and retention in the ER, Component of collagen biosynthesis
Reviewed in: Kim et al., 2009; Ellgaard and Ruddock, 2005; Appenzeller-Herzog and Ellgaard, 2008
Human ERp57 (P30101) 505; a-b-b’-a’
PLC component, Molecular marker of immunogenicity
Human PDIp (Q13087) 525; a-b-b’-a’
Peptide binding, Neurodegeneration
Human ERp72 (P13667) 645; a
o-a-b-b’-a’
ER retention
Human P5 (Q15084) 440; a
o-a-b
Tumor immune evasion
Human ERp18 (O95881) 172; a
Apoptosis against ER stress
Human ERp44 (Q9BS26) 406;a-b-b’
Protein retention in ER, Regulation of IP3R1 activity
TMX (Q9H3N1) /280; a
Reduction of ER stress
hPDI and hP5
The anti-cancer drug Vincristine inhibit the chaperone activity of hPDI and hP5
Human ERdj5 (Q8IXB1) 793; a’’-b-a
o-a-a’
ER-localised ERDj5 is member of the complex that recognizes and unfolds proteins for efficient retrotranslocation
Ushioda et al., 2008; Ellgaard and Ruddock, 2005;
Recombined PDIII, PDI (In vivo) n/a
Regulation of E2A transcription factor dimerisation and the development of the B lymphocyte lineage
Markus and Benezra, 1999
hsQSOX1 (HSU97276) Trx, ERV1-like
Regulation of growth through its role in the
formation of the extracellular matrix. Reviewed in, Thorpe et al.,2002
Chicken/ n/a retention of a protein in the endoplasmic reticulum
Parkkonen et al.,1988
In vitro n/a
Assist folding of denatured and reduced disulfide-containing proteins
Yao et al., 1997
Dog Microsomes n/a
Correct folding and assembly protein during biosynthesis (in vitro synthesis of wheat γ-gliadin7).
Bulleid et al., 1988
yeast pdi1/ n/a Secretion machinery and stress regulation Gasser et al., 2007
35
1.6 Properties and functions of plant PDIL
The PDIL families in plants are multi-gene, comprising 22 members in Arabidopsis, 19
in rice and 22 in maize. The Arabidopsis PDILs are from EST (expressed sequence tag)
and genomic sequence databases, and PDILs in rice and maize are from EST databases.
All plants PDILs contain 1 or 2 active sites. The PDIL family of Arabidopsis has been
reviewed (Houston et al., 2005); some relevant points are summarised below due to
relevance to this project.
Six of the thirteen PDIs (AtPDI1, AtPDI2, AtPDI3, AtPDI4, AtPDI5, AtPDI6;
nomenclature as per Lu and Christopher, 2008a; or AtPDIL1-3, AtPDIL1-4, AtPDIL1-
5, AtPDIL1-6, AtPDIL1-1 and AtPDIL1-2, respectively; Houston et al., 2005) contain
four domains (a, b, b’, a’) with an N-terminal signal peptide (SP) and a C-terminal
KDEL for ER retention/retrieval. One isoform (AtPDI8 or AtPDIL5-2) contains three
domains (a, b, b’) with a SP and a C-terminal transmembrane domain (TMD) and three
(AtPDI9, AtPDI10, AtPDI1; or AtPDIL2-3, AtPDIL2-2, AtPDIL2-1) contain SP, a and
a’. AtPDI7 and AtPDI12 (AtPDIL5-4 and AtPDIL5-3) contain only the a-domain, with
TMD at both N- and C-termini. AtPDIL5-1 contains a domain but others are
unconfirmed (Houston et al., 2005). Certain pairs (AtPDI1-AtPDI2, AtPDI3-AtPDI4,
AtPDI5-AtPDI6, AtPDI7-AtPDI12, AtPDI9-AtPDI10) share 54-79% identity at amino
acid level and phylogenetic trees suggest possible origins through duplications (Lu and
Christopher, 2008a).
In terms of functions, the genes encoding certain PDIs (AtPDI1, AtPDI5, AtPDI6,
AtPDI9, AtPDI10, AtPDI11) are upregulated in response to ER stress induced by
treating seedlings with chemicals (Lu and Christopher, 2008a). PDI5 is found to be
required for normal seed development through regulation of the timing of PCD
(programmed cell death) by chaperoning and inhibiting Cys proteases (Farquharson,
2008; Ondzighi et al., 2008). Likewise, PDIL2-1 functions in embryo sac maturation
and pollen tube guidance, through its roles in maturation of secreted or plasma
membrane proteins (Wang et al., 2008). Two PDIs (PDI-52 and PDI-65, Lu and
Christopher, 2006; likely to be AtPDIL1-1 and/or AtPDIL1-2, and AtPDIL1-4, Houston
et al., 2005) are identified to be chloroplast localised, and PDI-52 interacts with other
36
proteins to form a 220kDa complex to regulate enzymes associated with starch
metabolism (Lu and Christopher, 2006; 2008b).
Three of the seven APRLs (PRH-19, PRH-26, PRH-43, Gutierrez-Marcos et al., 1996;
or AtAPR1, AtARP3, AtAPR2, Houston et al., 2005) have chloroplast transit peptides
with the motif VHVA, VSAA or VHLA, similar to the cleavage site of chloroplast SP,
V/A-X-A/C↓A (Gavela and von Heijine, 1990). PRH-19 shows the PDI domain with
WCPFC (Gutierrez-Marcos et al., 1996). The AtQSOX1 and AtQSOX2 contain an N-
terminal SP, the a/a’ domain (YAV--KVA) with WCPAC, and an ERV1 domain
(DFV/I--CYL) (Thorpe et al., 2002). The key functions of Arabidopsis PDILs and their
orthologues (if known) are summarised in Table 1.5.
Table 1.5 Properties and functions of PDIL members in Arabidopsis and their orthologues in other plants
Namea, Genomic locus Orthologues in other plants/human Key functions,
reference
AtPDI5 (AtPDIL1-1); At1g21750 Required for proper seed development, regulates timing of PCD by chaperoning and inhibiting Cys proteases (Ondzighi et al., 2008; Lu and Christopher, 2008a)
AtPDIL1-2 (AtPDI6); At1g77510 GmPDIL-1 (soybean); Os04g35600 (rice); AJ277377 (wheat) GmPDIL-2 (soybean); Os02g01010 (rice) GmPDIL-1 and GmPDIL-2 play a role in folding of storage protein by both thiol-oxidoreductase and chaperone activities (Kamauchi et al., 2008; Lu and Christopher, 2008a)
AtPDIL1-3 (AtPDI1); At3g54960
AtPDIL1-4; (AtPDI2; PDI-65) At5g60640
Assisting with regulation of enzymes associated with starch metabolism (Lu and Christopher, 2006, 2008b)
AtPDIL1-5 (AtPDI3); At1g52260 n/a; Houston et al., 2005
AtPDIL1-6 (AtPDI4); At3g16110 n/a; Houston et al., 2005
AtPDIl2-1 (AtPDI11); At2g47470
Function in embryo sac development, ovule structure, pollen tube guidance, through roles in maturation of secreted or plasma membrane proteins in Arabidopsis. (Wang et al., 2008; Lu and Christopher, 2008a)
AtPDIL2-2 (AtPDI10); At1g04980 AtPDIL2-3 (AtPDI9); At2g32920
GmPDIM (soybean); Os09g27830 (rice). GmPDIM plays a role in folding of storage proteins as both the thiol-oxidoreductase and chaperone; responsible for ER-stress response (Kamauchi et al., 2008; Lu and Christopher, 2008a)
AtPDIL5-1; At1g07960 n/a. Houston et al., 2005 AtPDIL5-2 (AtPDI8); At1g35620 n/a Houston et al., 2005 AtPDIL5-3 (AtPDI12); At3g20560 n/a Houston et al., 2005 AtPDIL5-4 (AtPDI7) At4g27080 n/a Houston et al., 2005
AtAPR1 (PRH-19); At4g04610 AtAPR2 (PRH-43); At1g62180 AtAPR3 (PRH-26); At4g21990
Gutierrez-Marcos et al., 1996
AtQSOX1; At1g15020, AtQSOX2; At2g01270
HsQSOXl in regulation of growth by its requirement for the formation of the extra-cellular matrix (Thorpe et al., 2002)
aPDIL names as per Houston et al., 2005; other names given in parenthesis and the corresponding
authors given in column 2. n/a; orthologs in other species not known or confirmed.
Seven PDIL members in soybean (Glycine max) are known so far. Two members (60
and 63 kDa) were first purified from soybean root and stem, respectively; the 63 kDa
37
PDI presented its enzyme activity in a complex of 120kDa (Shorrosh et al., 1993;
Kainuma et al., 1995). The cDNAs of GmPDIL-1, GmPDIL1-2, GmPDIM, GmPDIS-1
and GmPDIs-2, isolated from leaves and expressed in E. coli, showed thiol-
oxidoreductase activity (Kamauchi et al., 2008; Wadahama et al., 2008). The genomic
sequence (10 exon structure) of GmPDIl-1 is most similar to AtPDIL1-2, a rice (Oryza
sativa) PDI (encoded at locus Os04g35600) and wheat (AJ277377); GmPDIL-2 (12
exons) is most similar to AtPDIL1-3 and product of Os02g01010, and GmPDIM is most
similar to AtPDIL2-2 and AtPDIL2-3 and product of Os09g27830. The structure of
GmPDIM is similar to human P5 (see section 1.5.5) and is up-regulated under ER-stress
during normal seed maturation. GmPDIL-1, GmPDIL1-2 and GmPDIM are localised in
the ER lumen and play a role in the folding of storage proteins through both thiol-
oxidoreductase and chaperone activities (Kamauchi et al., 2008; Wadahama et al.,
2008).
A few other plant PDILs have been reported. The barley (Hordeum vulgare) PDI
protein levels correlate with rate of hordein accumulation, implying participation in
formation of disulfide bonds in hordeins (Mogelsvang and Simpson, 1998). The
expression of HvPDI is abundant in the caryopsis but weak in other tissues (pericarp,
stems, leaves), also supporting a role in assembly of storage proteins (Kim et al., 2008).
Expression of PDI in leaf tissue of spinach (Spinacia oleracea) induced by light
exposure shows a similiar pattern to ER luminal Hsp70, indicating coordinate light/dark
regulation for molecular chaperone (Li and Guy, 2001). The Alfalfa P5 contains two a-
like domains (ao, a,) (Ferrari and Soling, 1999). The cDNA of SPPDI1 of sweet potato
encodes a putative protein of 503 aa with two active sites and C-terminal KDEL, the
expressed protein exhibits a glutathione-insulin trans-hydrogenase activity, and the high
expression of SPPDI1 in storage roots indicates a function in coping with specific tissue
development (Huang et al., 2005). The putative product of OaPDI of Oldenlandia
affinis (Rubiaceae) shows domains a, b, b’ and a’, and interaction of the expressed
protein demonstrates it is involved in the biosynthesis of the insecticidal cyclotides
(knotted circular proteins), mainly through its chaperone activity by noncovalent
interactions (Gruber et al., 2007; 2009). Twelve PDIs (plus 1 QSOXL and 6 APRL)
have been identified from rice (Oryza sativa) EST database (Houston et al., 2005). Of
38
these, OsPDIL1-1 (LOC_Os11g09280) has a very important role in storage protein
regulation (detailed in section 1.7).
Although wheat is the world’s largest crop plant, molecular work on wheat PDI has
been much more limited than the corresponding work in rice. Three cDNA clones
isolated in M. Bhave’s group from the T. aestivum endosperm library encode putative
proteins of 513-516 aa (Johnson et al., 2001), the gene structures show 10 exons
(Johnson et al., 2004a) and are located on chromosomes 4AL, 4BS, and 4DS (Ciaffi, M
et al. 1999). Their putative orthologue in rice (LOCOs_11g09280) is close to the esp2
locus, indicating the quantitative trait loci related to wheat storage protein deposition
may involve PDI loci (Johnson and Bhave, 2004a; Johnson et al.,
2006). The isolated promoter sequences of three PDI genes on chromosomes 4A, 4B,
and 4D show cis-acting regulatory elements for endosperm specific regulation (Ciaffi et
al., 2006).
39
Table 1.6: PDI properties and functions in plants (excluding Arabidopsis)
Species/ tissues
(Location) Size Function and other properties Reference
Soybean/root and stem
60 kDa Expression increase (protein level) in developing seeds
Shorrosh et al., 1993
Soybean 63 kDa Native enzyme has a molecular weight of 120 kDa, active sites APWCGHCK
Kainuma et al., 1995
Soybean/leaves 525aa, 551 aa, 438 aa
GmPDIL-1, GmPDIL-2, and GmPDIM have function in folding of storage protein; GmPDIM is up-regulated under ER-stress
Kamauchi et al., 2008; 2007
Sweet potato 503 aa SPPDI1 has function in coping with particular tissue development
Huang et al., 2005
Coffee plant n/a OaPDI can biosynthesis of cyclotides by noncovalent interactions
Gruber et al., 2007; 2009
Barley (Hordeum vulgare)/(ER)
60 kDa Participate in the formation of disulfide bonds in B-, D- and γ- hordeins
Mogelsvang et al., 1998
Barley/HvPDI 507 aa Assembly seed storage proteins Kim et al., 2008
Maize/ endosperm development (ER)
n/a Increase of PDI expression in floury-2 endosperm: molecular chaperone: zein processing and assembly into protein bodies.
Li and Larkins, 1996
Rice (Oryza sativa)/ Esp 2 mutant
n/a Essential role in the segregation of proglutelin and prolamin polypeptides within the ER lumen.
Takemoto et al., 2002
Rice (Oryza sativa)/ Endosperm
n/a ER chaperones and disulfide bonds formed at the dicysteine residues in CCxGL play critical role in sorting fused protein in the endosperm cells.
Kawagoe, Y. et al. 2005
Wheat/Endosperm/(ER)
n/a Putative: formation of disulphide bonds during biosynthesis of wheat storage proteins.
Roden et al., 1982
Dog/Microsomes/ Pancreas/(ER)
n/a Correct folding and assembly of proteins during biosynthesis (in vitro synthesis of wheat γ-gliadin7)
Bulleid and Freedman, 1988
Wheat/Caryopses /ER
n/a Transcripts for BiP, PDI and PPI are highest at earlier stages of development
Grimwade et al., 1996
Wheat/Endosperm
n/a Storage protein folding and accumulation in the ER
Dupont et al., 1998
Wheat n/a Play role in plant defense response to pathogen
Ray et al., 2003
1.7 Evidence for roles of ER lumen ‘foldase’ enzymes and chaperones
in storage protein folding and deposition
The processes of mature storage protein folding, assortment and deposition occur in the
ER, as summarized above (section 1.2.4). During protein folding, hydrophobic
residues can form undesirable interactions within or between polypeptide chains. With
the aid of enzymes and/or chaperones, protein folding can be performed properly
without the risk of aggregation caused by misfolding. Three groups of proteins are
likely involved: the Binding Protein (BiP)/heat shock protein (Hsp70) chaperones
(Galili et al., 1998), and the enzymes PPIases and PDI (Shewry et al., 1995; Boston et
al., 1996; Dupont et al., 1998).
40
In support of this expectation, BiP has been shown to be present within PBs in wheat
endosperm at 13 and 16 DAA of development by immunogold labeling (Levanony et
al., 1992). BiP also increases dramatically in the PBs in the ER of the maize mutant
floury-2 which produces abnormal storage proteins (Li and Larkins, 1996), and is
induced under ER stress in maize with assembly-defective storage proteins (Wrobel et
al., 1997). Interaction of prolamins with BiP in the formation of ER-derived PBs has
been confirmed experimentally in transgenic rice (Saito et al., 2009).
PPIases have the key function of catalysing the cis-trans isomerisation of the peptide
bond preceding proline residues, a rate-limiting step in the folding of newly synthesised
proteins (Galat, 1993). Thus PPIases are expected to be directly relevant to the folding
processes of the proline-rich wheat prolamins, either themselves and/or in cooperation
with other chaperones (BiP and PDI). Of the three major classes of PPIases (described
in 1.3.2), Cyp members have been indicated to be ER-localised in maize (Sheldon et al.,
1996) and Arabidopsis (Saito et al., 1999b; He et al., 2004; Romano et al., 2004a).
The foldase enzyme PDI has the key activity of catalysing disulfide bond breakages,
formation or rearrangement (Creighton et al., 1980; Schmid et al., 1986) as detailed
above, and thus is highly likely to play direct roles as a foldase (or as a chaperone) in
the folding of wheat prolamins. In support of this, PDI has been shown to be
sequestered in wheat PBs (Roden et al., 1982) and identified in the ER lumen and co-
localised with the storage proteins in the dense PBs (Shimoni et al., 1995b). Messenger
RNAs of BiP and PDI have been reported to accumulate to maximal levels in the
middle stages (specifically 5 to 14 days post-anthesis) of wheat endosperm development
(Dupont et al., 1998) and the two proteins are suggested to act together in storage
protein deposition in maize (Li and Larkins, 1996) and barley (Mogelsvang and
Simpson, 1998). The critical role of PDI in protein folding was established through
studies of the rice mutant esp2 endosperm storage protein 2) by Takemoto et al. (2002).
Rice storage proteins are mainly divided into prolamins and glutelins, accumulating into
two types of protein bodies, PB-I and PB-II, respectively. PB-I are relatively small and
spherical, while PB-II are large and irregularly shaped. The mutant esp2 contained
normal PB-II with mainly glutelins, but lacked the normally spherical PB-I and instead
had a new type of PB which was smaller than PB-I, had polyribosomes attached to its
41
surface indicating it was generated directly from the ER, and most interestingly, it
contained both glutelin and prolamin polypeptides. Three ER lumenal chaperones (BiP,
calnexin, and PDI) are present in the wild type rice, but esp2 was lacking in PDI
transcripts, establishing that PDI has an essential role in disulfide interchange reactions
and assortment of the prolamin and proglutelin polypeptides (Takemoto et al., 2002).
The sequences in prolamins and proglutelins that promote interactions with PDI are
shown to be LxxC, CCxQL, and PxxC (Kawagoe et al., 2005).
There is also substantial circumstantial evidence for role of PDI in wheat storage protein
folding processes. PDI was initially identified in mature endosperm in wheat and
suggested to be involved in formation of disulphide bonds during wheat storage protein
processing (Grynberg et al., 1977; Roden et al., 1982). The role of PDI in correct
folding and assembling of storage proteins was demonstrated through wheat γ-gliadin7
synthesis in vitro by PDI from microsomes in dog (Bulleid and Freedman, 1988). PDI
was identified in the lumen of ER and co-localised with storage proteins in the dense
protein bodies in wheat (Shimoni et al., 1995b). Expression of PDI and BiP were noted
to be relatively high at both the mRNA and protein levels at the early seed development
stages (Grimwade et al., 1996; DuPont et al., 1998). The highly expressed genes in
wheat endosperm also include the gene (BE438375) encoding protein disulfide
isomerase 3 (PDI3), through the wheat endosperm transcriptome analysis (Laudencia et
al., 2006). Additionally, PDI possibly functions in defense from pathogens (fungi) in
wheat (Ray et al., 2003).
1.8 Summary of the above literature and aims of this project
The quality and quantity of storage proteins in the wheat endosperm are important due
to wheat being one of the world’s most important crops. These proteins are commonly
grouped into the monomeric gliadins and polymeric glutenins, which form a highly
structured protein network in the dough that determines its viscoelastic properties and
end-uses. Genetic differences as well as environment stresses such as high temperature
and lack of water are known to influence the quality and quantity of these proteins. The
processes of cereal grain storage protein folding, resolution of aggregates, assortment of
the various subunits and deposition by the two main routes as well as the ER stress
created in this process, and responses to external abiotic stresses all strongly point to an
42
involvement of ‘foldase’ enzymes in their enzymatic and/or chaperone roles. The
cyclophilin and protein disulphide isomerase gene families represent two ideal
candidates for not only these functions, but also potentially for additional roles
suggested by literature on other organisms. However, there is limited information on
these gene families in two of the world’s largest cereal crops, wheat and rice, warranting
further investigations.
1.8.1 Aims of this project
The major aim of this project was to identify the cyclophilin (Cyp) and protein
disulphide isomerase (PDI)-like superfamilies in wheat and rice by informatics tools,
conduct their comparative molecular and phylogenetic analysis, and identify any
previously uncharacterised isoforms with potentially important roles in grain quality or
plant development processes. Another aim of this project was to experimentally isolate
and characterise the genes encoding putative ER-localised cyclophilin B proteins in
wheat, to physically and genetically map these genes, and to identify the putative
promoter sequences of the CypB genes in wheat and rice. The specific aims were as
follows.
• To identify and analyse all potential genes encoding Cyp families in rice and wheat
by use of genomic and expressed sequence databases and bioinformatics tools.
• To use the data of these Cyp genes to predict the putative proteins and analyse their
important features through bioinformatics analyses and comparisons to the
Arabidopsis and/or human isoforms.
• To similarly identify all potential members of PDIL gene families in rice and wheat.
• To similarly predict and analyse all putative PDIL proteins in order to identify new
members of relevance to any grain quality traits.
• To use information associated with above rice Cyp and PDIL loci to predict the
physical locations of wheat genes if possible.
• To amplify, clone and sequence CypB genomic genes from common wheat, and its
tetraploid and diploid progenitors.
• To amplify, clone and sequence CypB cDNAs from common wheat.
• To characterise the putative CypB proteins from above species by bioinformatics
analysis.
43
• To assign the CypB genes to different genomes through comparison of gene
structures and sequence data from hexaploid, tetraploid and diploid wheats.
• To identify the CypB gene in rice by bioinformatics analysis of its genome data.
• To compare the important structural and functional features of the putative CypB
proteins in wheat, rice and other orthologues to their function and relatedness.
• To clone and sequence the putative promoters of CypB gene in wheat, compare
with that of the rice CypB gene, and identify any unique regulatory elements.
• To use any differences in the three homeologous CypB genes in wheat to develop
molecular markers to physically map the genes using nullisomic-tetrasomic and
deletion lines of wheat. Also to predict the physical location of wheat CypB genes
using information associated with the rice CypB locus.
• To use the data of the three homeologous CypB genes to amplify, restriction map
and/or sequence the genes from ten parental lines used for genetic mapping.
• To apply any differences in CypB in the parental lines to genetically map CypB
genes using the DH progeny mapping lines of the relevant crosses.
• To identify from available information any QTLs of interest occurring in the general
area of the CypB and PDIL loci, on the respective chromosomes of rice and wheat.
• To conduct preliminary analysis of the expression of PDI ad ER-localised Cyps in
different tissues and developing grains in wheat.
44
Chapter 2
MATERIALS AND METHODS
45
2 Materials and Methods
2.1 Commercial kits, materials and solutions
Table 2.1 Commercial kit and reagents used during this project
Kit/material/solution Supplier Purpose
HaeIII (BsuRI) (GG↓CC; assay temperature: 37
oC; Buffer R)
Fermentas, Ontario, Canada
Generation of RFLPs, allele specific primers confirmation, physical mapping
NcoI (C↓CATGG; assay temperature: 37oC;
NEBuffer 3) New England Biolabs, (NEB), Madison, USA
Generation of RFLP for physical mapping
HpyCH4IV (A↓CGT; assay temperature: 37
oC; NEBuffer 1)
Generation of RFLP for genetic mapping
SacI (GAGCT↓C; assay temperature: 37oC;
NEB Buffer 1) Generation of ligated circles of DNA for inverse PCR
Wizard® Genomic DNA purification kit
(contains nuclei lysis solution, protein precipitation solution, DNA rehydration solution)
Promega, Australia
Plant genomic DNA isolation
Wizard®
Plus Minipreps DNA Purification System (contains cell suspension solutions, cell lysis solution, neutralization solution, column wash solution, minicolumns, alkaline protease solution)
Plasmid DNA purification
pGEM-T Easy Vector System (contains pGEM-T Easy Vector, T4 DNA ligase, 2X ligation buffer)
TA Cloning of PCR products
Perfectprep®
Gel Cleanup kit (contains binding buffer, wash buffer, elution buffer, spin columns)
Eppendorf, Hamburg, Germany
Purification of DNA from gel
TRIsureTM
Bioline, London, UK
Total RNA isolation BioScript
TM Reverse transcription
RNase Inhibitor Prevention of RNAase activity
Biomix (2X) (contains BIOTAQTM
DNA polymerase, (NH4)2SO4, Tris-HCl, Tween 20, dNTPs, MgCl2)
PCR
10 X PCR buffer BIOTAQ
TMDNA Polymerase
dNTP set (4X25 µmol)
GeneRulerTM DNA Ladder Mix Fermentas, Ontario, Canada
Electrophoresis Lambda DNA/EcoRI + HindIII Marker
Ethidium bromide (10 mg/mL) Sigma
BDT (Big Dye Terminator) v3.1 Ready Mix Applied Biosystems, Australia
DNA sequencing
2.2 Prepared solutions
All solutions listed in Table 2.3 were prepared using established protocols (Sambrook et
al., 2001). The solutions were sterilised by autoclaving (121oC for 20 minutes), or
filtering through a 0.45 µm MF-Millipore syringe filter (Millipore, Madison, USA)
under vacuum.
46
Table 2.2 Prepared solutions, media and plates
Solution Preparation
Transformation and cloning
Luria-Bertani plates 7.5 g agar in 500 mL of LB medium TfbI Per 200 mL: 0.588 g potassium acetate (30 mM), 2.42 g rubidium
chloride (100 mM), 0.294 g calcium chloride (10 mM), 2.0 g manganese chloride (50 mM), 30 ml glycerol (15% v/v)
TfbII Per 100 mL: 021 g MOP (10 mM), 1.1 g calcium chloride (75 mM), 0.121 g rubidium chloride (10 mM), 15 mL glycerol (15% v/v)
Psi broth Per litre: 5 g Bacto yeast extract, 20 g Bacto Tryptone, 5 g magnesium sulfate, Adjust pH to 7.6 with 1 N potassium hydroxide
X-Gal 20 mg/mL: 20 mg (5-bromo-4-chloro-3-indolyl-β-D-galactoside) in 1 mL N,N’-dimethyl-formamide, filter sterilized.
IPTG 0.1 M: 1.2 g (Isopropylthio-β-D-galactoside) in dH2O in 50 mL, filter-sterilised
Ampicillin 20 mg/mL: 20 mg in dH2O in 1mL, filter sterilized
Luria-Bertani plates with X-Gal
7.5 g agar in 500 mL of LB medium, with 100 µg/mL ampicillin, 0.5 mM IPTG, 80 µg/mL X-Gal.
2xTY medium Per litre: 16 g Tryptone, 10 g Yeast extract, 5 g NaCl, adjust pH to 7.0 with 5 N NaOH.
Luria-Bertani (LB) medium
Per litre: 10 g Bacto-tryptone, 5 g Bacto-yeast extract, 5 g NaCl, adjust pH to 7.0 with 5 N NaOH.
Glycerol 60% (v/v) DNA and RNA preparation, PCR, electrophoresis, and DNA sequencing
70% ethanol Per 10 mL: 3 mL ethanol, 7 mL dH2O.
Chloroform/isoamyl-alcohol
Chloroform: isoamylalcohol: 24:1 (v/v)
Isopropyl alcohol 100%
RNase A (10 mg/mL) 0.01 M sodium acetate, 0.1% (v/v) Tris-HCl (1 M, pH 7.4). 6x Loading Dye 0.25% Bromophenol blue (w/v), 0.25% Xylene Cyanol FF (w/v), 40%
Sucrose (w/v)
TAE Buffer 40 mM Tris-acetate, 1mM EDTA 1 x ligation buffer 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP
5 x BDT buffer 400 mM Tris pH9.0; 10 mM MgCl2
0.2 mM MgSO4 3 ml MilliQ water, 7 ml Ethanol, 2 µl 1 M MgSO4
2.3 Equipment Table 2.3 Equipment used during this project
Equipment Manufacturer Purpose
Humidity cabinet Thermoline, Coburg, Australia Growth of wheat seedlings
Electrophoresis power supply-EPS301 Minnie Gel Unit
General Electric (GE) Healthcare, Buckinghamshire, UK
Electrophoresis
Apollo large gel unit Apollo mini gel tank
Continental Lab Products, California, USA
Electrophoresis
UV transilluminator Integrated Sciences, Chatswood, NSW, Australia
Agarose gel visualisation
GeneQuant pro UV/Vis Spectrophotometer
GE Healthcare, Buckinghamshire, UK
Quantification of DNA and RNA
C3040 digital camera Olympus, Tokyo, Japan Capturing images of gels
Bio-Rad Chemidoc XRS documentation station
Bio-Rad, California, USA Capturing images of gels or chemiluminescent blots
Mastercycler Eppendorf, Hamburg, Germany The Polymerase Chain reaction (PCR)
MyCyclerTM
Bio-Rad, California, USA PCR
Finnpipettes Thermo Electron, Madison, USA Dispensing liquids
47
2.4 Plant materials and DNA stocks used in this project
The seeds of wheat samples, listed in Tables 2.4 and 2.5, were kindly provided by Dr.
Greg Grimes, Australian Winter Cereals Collection (AWCC, Tamworth, NSW,
Australia). Genomic DNA was extracted from the nullisomic/tetrasomic (NT) lines and
kindly provided by Dr. Kerrie Forrest in our laboratory (now at Biosciences Research
Centre, Department of Primary Industry, Victoria, Australia). Genomic DNA samples
of ten deletion lines of chromosome 7 (Table 2.6) and 163 doubled haploid (DH)
progeny lines of the Tasman x Sunco cross (described later) were kindly provided by
Prof. Rudi Appeals at the Western Australia State Agriculture Biotechnology Centre
(SABC, Murdoch University, Western Australia, Australia).
Table 2.4 Wheat cultivars and progenitors
Aus Accession
Species Cultivars Genome Ploidy and chromosome no.
27032 T. urartu na AA 2n=2x=14
21927 Ae. tauschii na DD 2n=2x=14
26510 T. turgidum ssp. durum
na AABB 2n=4x=28
119 T. aestivum Chinese Spring AABBDD 2n=6x=42
99124 T. aestivum Rosella AABBDD 2n=6x=42
22660 T. aestivum Cranbrook AABBDD 2n=6x=42
11612 T. aestivum Halberd AABBDD 2n=6x=42
25557 T. aestivum Tasman AABBDD 2n=6x=42
23455 T. aestivum Sunco AABBDD 2n=6x=42
91167 T. aestivum CD87 AABBDD 2n=6x=42
91172 T. aestivum Katepwa AABBDD 2n=6x=42
91173 T. aestivum Kukri AABBDD 2n=6x=42
91174 T. aestivum Janz AABBDD 2n=6x=42
91177 T. aestivum Egret AABBDD 2n=6x=42
22177 T. aestivum Sunstar AABBDD 2n=6x=42
Table 2.5 Nullisomic/tetrasomic (NT) lines used for physical mapping
AUS Accession Line Chromosomes AUS Accession Line Chromosomes
91353 N1A-T1B 1 - BBBBDD 91375 N4D-T4A 4 - AAAABB 91356 N1B-T1D 1 - AADDDD 91377 N5A-T5B 5 - BBBBDD 91358 N1D-T1B 1 - AABBBB 91379 N5B-T5A 5 - AAAADD
91359 N2A-T2D 2 - AADDDD 91381 N5D-T5A 5 - AAAABB 91361 N2B-T2A 2 - AAAADD 91383 N6A-T6B 6 - BBBBDD
91363 N2D-T2A 2 - AAAABB 91385 N6B-T6A 6 - AAAADD 91366 N3A-T3D 3 - BBDDDD 91387 N6D-T6A 6 - AAAABB 91368 N3B-T3D 3 - AADDDD 91348 N7A-T7D 7 - BBDDDD
91370 N3D-T3B 3 - AABBBB 91350 N7B-T7D 7 - AADDDD 91372 N4A-T4D 4 - BBDDDD 91352 N7D-T7B 7 - AABBBB
91374 N4B-T4D 4 - AADDDD
48
Table 2.6 Deletion lines of chromosome 7 used for physical mapping*
Lines Deletion arms Deletion break point Lines Deletion
arms Deletion
break point
7AS-8 7AS 0.89 7DS-4 7DS 0.61 7AL-1 7A 0.39 7DS-5 7DS 0.36 7BS-1 7BS 0.27 7DL-2 7DL 0.61
7BL-2 7BL 0.33 7DL-3 7DL 0.82 7BL-7 7BL 0.63 7DL-5 7DL 0.30
*Generated from T. aestivum cv Chinese Spring. Deletion break-points: Endo and Gill, 1996 . (http://www.k-state.edu/wgrc/Germplasm/Deletions).
Methods
The Methods section contains two parts: the general molecular methods part outlines
the experimental work, with some essential bioinformatics. The bioinformatics
methods part outlines the computational tools used during this project.
General molecular methods
2.5 Growth of seedlings for DNA isolation
Seeds of the experimental variety Chinese Spring and eleven cultivars of Triticum
aestivum L. (AABBDD), the tetraploid durum wheat (T. turgidum ssp. durum, AABB)
and the diploid progenitors of wheat, T. urartu (AA) and Aegilops tauschii (DD) (as
listed in Table 2.4), were germinated and grown under controlled conditions (18-hour
day lengths, 70% humidity, 25°C temperature) for 8-12 days. Young leaves from
seedlings were snap-frozen in liquid nitrogen and used directly for genomic DNA
isolation or stored at -80oC for later isolations.
2.6 Preparation of genomic DNA
Genomic DNA (gDNA) was extracted from the frozen leaf tissue of bulked seedlings
(Rosella, Chinese Spring, and durum) or individual seedlings (T. urartu, Ae. tauschii,
and the ten cultivars used as parental lines in the Australian National Wheat Molecular
Marker Program (NWMMP) (Kammholz et al., 2001), i.e. Cranbrook, Halberd,
Tasman, Sunco, CD87, Katepwa, Janz, Kukri, Egret, Sunstar) (Table 2.4) using the
Wizard genomic DNA purification kit (Promega Australia). Leaf tissues (40 mg) were
kept frozen in liquid nitrogen and ground into fine powder using a 1.5 mL
microcentrifuge tube pestle. The Nuclei Lysis solution (600 µL) was added and
incubated for 15 minutes at 65oC. RNase solution (3 µL) was added to the cell lysate
49
and the sample incubated for 15 minutes at 37oC. Protein precipitation solution (200
µL) was added to the lysate and the sample was centrifuged for 15 minutes at 14.5 x
1000 rpm. The supernatant containing DNA was carefully transferred to another 1.5
mL microcentrifuge tube containing 600 µL isopropanol and centrifuged for 5 minutes.
The DNA pellet was washed with 600 µL of 70% ethanol, air-dried, dissolved in 100
µL sterilised distilled H2O (dH2O) and stored at -20oC. An aliquot was diluted and the
DNA concentration was determined by the GeneQuant Pro Spectrophotometer.
2.7 The polymerase chain reaction (PCR)
2.7.1 Synthesis of oligonucleotide primers
A Tentative Consensus (TC) sequence TC84720, obtained from the TaGI TC Report in
the TIGR wheat genome database (http://www.tigr.org/tdb/e2k1/tae1) had been
identified earlier as encoding a putative ER-localised cyclophilin B (Johnson and
Bhave, 2004b). This TC was later found to be split into TC264493 and TC264488.
TC264488 (963bp) was selected for the design of primers, and was available
(http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=wheat, accessed
04/2008) through the TIGR wheat genome database ‘DFCI Wheat Gene Index’.
TC264488 was later split into (TC284235, TC308497, TC340662, and TC316845)
(http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=wheat, last accessed
04/2009). The consensus sequence of primers, their positions on the TC264488, and
expected sizes based on TC264488 are shown in Table 2.7 and Appendix I. The
primers were used for amplification of CypB genes from gDNA of T. aestivum cv.
Rosella and its progenitors including the durum wheat and the diploid progenitors T.
urartu (AA) and Aegilops tauschii (DD), and cDNA of T. aestivum cv. Cranbrook.
2.7.2 Typical PCR conditions
All PCR amplifications were conducted using 200 ng gDNA or 100 ng first-strand
cDNA (introduced later), 1 µL of each primer (0.1µg /µL) and 25 µL of 2X BioMix, in
50 µL volumes. The PCR conditions were initial denaturation of template DNA at
95oC for 5 mins, followed by 35 cycles of denaturation (94oC, 45 seconds), primer
annealing (at temperatures shown in Table 1, 45 seconds) and extension (72oC, 3
minutes), then a final extension (72oC, 5 minutes).
50
Table 2.7 Primer pairs used for PCR or IPCR amplifications and/or sequencing of CypB gene and its promoter, physical mapping and genetic mapping
Forward primer Reverse primer ATc EL* Purpose
WC1 (114-131bp)a
5’ATACGATCCAAGATGGCG 3’ CYPBR5 (411-430 bp)
a
5’ CTGTGGAACGAACTCCCCTT 3’ 57
oC
317bp
Fragment (WC1-CYPBR5)
WC4 (253-271bp)a
5’CGCACAAGGTCTACTTCGA3’ CYPBR1 (731-748 bp)
a
5’CTGTCGGCAATGACAACC3’ 54
oC ~1800bp
Fragment (WC4-CYPBR1)
WC1 (114-131bp)a
5’ATACGATCCAAGATGGCG 3’ CYPBR9 (943-960 bp)
a
5’ ACGGTGGTGTGTGATGCC3’ 57
oC ~2300bp
Fragment (WC1-CYPBR9)
WC1 (114-131bp)a
5’ATACGATCCAAGATGGCG 3’ CYPBR9 (943-960 bp)
a
5’ ACGGTGGTGTGTGATGCC3’ 57
oC ~850bp
cDNA Fragment
CYPBF3 (366-386bp)a
5’ACAGGTGAGAAAGGCATGGGC
CYPBIPCR-F4 (84-98)b
5’AGGGTGGCGGCGACG3’ 62
oC N/A
IPCR CYPBF3 (366-386bp)
a
5’ACAGGTGAGAAAGGCATGGGC
CYPBIPCR-R1 (126-143 bp)a
5’CCACGCCCTCATCGCCAT3’ 62
oC N/A
T7 5’GTAATACGACTCACTATAGGGC
SP6 5’TATTTAGGTGACACTATAGAAT3’
45oC Clone-dependent
WC3 (299-316bp)a
5’TCGGGTTGTCATGGGACT3’ CYPBR4 (255-276 bp)
a
5’CGATGTCGAAGTAGACCTTGTG3’
Internal sequencing for CypB gene
CYPB IPCR-F2 5’GCGTAGTAGATCAGTAGTAGC3’
CYPB IPCR-R2 5’GTGTCGTGGTATTGAA3’
Internal sequencing for CypB promoter
WC3 (299-316bp)a 5’TCGGGTTGTCATGGGACT3’
CYPBR5 (411-430 bp)a 5’ CTGTGGAACGAACTCCCCTT 3’
55o
~1000bp
Physical Mapping
CYPB-A1(625-643bp)b
CCTGCACTGCACCTAATCA CYPBR-A1 (1737-1754 bp)
b
5’CTT GCC GCT GTT GCC CAT3’
57o
1130bp
Genetic mapping (A genome)
CYPBF4 (84-98bp)b
5’CGTCGCCGCCACCCT3’ CYPBR-B1(1770-1789 bp)
b
5’ GGCATGGTCAGCAACCAGTG3’
62o
1706bp
Genetic mapping (B genome)
CYPB-D1 (502-519bp)b
5’CCAATATCGCATGCCTGT 3’ CYPBR-D1(1730-1750 bp)
b 5' GATTAAGTGGGGCATGGTCAG3'
54o
1249bp
Genetic mapping (D genome)
a
Primer positions are based on the TC264488. b
Primers positions are based on the genomic sequence data obtained during this project. Bold red letters in the allele specific primers indicate positions specific to certain genomes.
cAT: annealing temperature used for the pair.
*EL (expected length): refers to estimated minimum lengths of gene sections: (i) length of fragment WC1-CYPBR5 predicted based on sequence of TC264488; (ii) length of fragment WC4-CYPBR1 based on TC264488 and experimentally determined length of fragments WC1-CYPBR5; and (iii) length of fragment WC1-CYPBR9 based on TC264488 and the experimentally determined lengths of fragment WC1-CYPBR5 and WC4-CYPBR1; (iv) length of cDNA fragment based on TC264488; (v) length of fragment CYPBF3-CYPBR1 based on sequence data obtained for fragment WC1-CYPBR9 from T. aestivum cv. Rosella. Size of inverse PCR (IPCR) products cannot be estimated (N/A). Expected size of fragment for physical mapping and genetic mapping were based on sequence data obtained for fragment WC1-CYPBR5. Bold and underlined ATG in the WC1 primer sequence is the start codon.
51
2.8 Agarose gel electrophoresis and purification of PCR products
Aliquots (5 µL) of PCR products were typically electrophoresed on 1% agarose gels, at
100V for 40-60 mins, to determine their sizes. The remaining 45 µL of PCR products
of interest were purified with Perfectprep® Gel Cleanup Kit (Eppendorf) in order to
remove excess primers and PCR reagents. Briefly, the PCR product was resolved on
1% agarose gels, the gel slice was excised and weighed. Three volumes of binding
buffer were added for every volume of gel slice (1µg: 1µL) and incubated at 50oC for
10 mins. One volume of isopropanol was then added to the liquefied gel slice and
mixed. The sample (800 µL) was added to the spin column and centrifuged at 14.5 x
1000 g for 1 min, washed with 750 µL of diluted wash buffer and collected in 30 µL
dH2O.
2.9 Cloning of PCR products
2.9.1 Preparation of transformation-competent E. coli JM109
The stored E. coli JM109 (-70oC) culture was streaked on an LB agar plate and grown
overnight at 37oC, then a single colony was inoculated into 3 mL LB broth and grown
overnight at the same temperature. One mL of this culture was transferred into 100 mL
Psi broth and incubated at 37oC until the cell density reached A550=0.48 (by shaking for
two hours then checking the absorbance every 30 mins). The Psi broth with competent
cells was held on ice for 15 mins and centrifuged for 5 mins at 5000 rpm. The
supernatant was discarded, 0.4 volume (of original volume) TfbI was added, cells
resuspended and held on ice for 15 mins. The cells were then pelleted for 5 mins at
5000 rpm, the supernatant discarded and the cells resuspended again in 0.04 volume (of
original volume) TfbII and held on ice for 15 mins. Finally, the cell suspension was
then distributed into 100 µL aliquots in 1.5 mL tubes and either used directly or stored
at -70oC.
2.9.2 Cloning of genomic copies or cDNA of genes
pGEM-T Easy Vector System kit (Promega) and the associated protocol were used to
clone the various PCR products generated from gDNAs or CDNAs (the latter is
explained below) from different wheat species. Ligation reactions (10 µL) contained
purified PCR products (3 µL), pGEM-T Easy Vector with T overhangs (1 µL), 2 x
Rapid Ligation Buffer (5 µL) and T4 DNA ligase 1 µL (3 units/µL) and were conducted
52
overnight at 4oC. The reaction mixes were then transferred to 100 µL JM109 competent
cells, held on ice for 40-60 mins, heat-shocked for 3 mins at 42oC and again held on ice
for 2 mins. 500 µL of 2xTY medium was added and the culture shaken at 37oC for 2
hours. 100 µL of the culture was plated onto LB plates containing AMP/IPTG/X-Gal
and incubated at 37oC overnight. White colonies (expected to contain recombinant
plasmids) were used for plasmid isolations.
2.10 Plasmid DNA isolation and RFLP analysis of polymorphism
For obtaining clones of the PCR product, a few (4-8) randomly selected white colonies
from its ligation/transformation (see above) were inoculated individually into tubes
containing 3 mL LB medium with ampicillin and incubated overnight at 37oC with
shaking. The cells were pelleted by centrifugation at 14.5 x 1000 g for 5 min and
plasmid DNA isolated by Wizard® Plus SV Minipreps DNA purification system
(Promega) with some modifications. Resuspension solution (250 µL) was added into
the microcentrifuge tube containing the pellet and the cells resuspended by vortexing.
Cell lysis solution (250 µL) was added and mixed by inverting the tube. Alkaline
protease solution (10 µL) was then added, mixed by inverting the tube and incubated for
5 minutes at room temperature. Neutralisation solution (350 µL) was added and mixed
by inverting. The sample was centrifuged at 14.5 x 1000 rpm for 10 mins at room
temperature. In order to avoid transferring any white precipitate (cell debris) with the
supernatant, the alkaline protease was added to the sample (in the same tube) and the
sample was centrifuged again. The clear lysate (approximately 850 µL) was transferred
to the prepared spin column and centrifuged at 14.5 x 1000 rpm for 1 min. The spin
column with the bound plasmid DNA was washed with 750 µL, then 250 µL washing
solution and the plasmid DNA eluted into a microcentrifuge tube with 100 µL dH2O.
Based on the predicted restriction sites in TC264488 (Appendix I), HaeIII (BsuRI) was
used for preliminary analysis of sequence variability in the gDNA clones. The inserts
from clones of interest were amplified using vector-based primers T7 and SP6 (Table
2.7) with the PCR protocol given above (section 2.7.2). The PCR products (8.5 µL)
were digested with 0.5 µL HaeIII (10 u/µL) in 10 µL and analysed by electrophoresis
on 1.7% gels.
53
2.11 DNA sequencing
2.11.1 DNA sequencing reactions
The sequencing was carried out using the dideoxy chain termination method, as per the
instructions of AGRF (Australian Genome Research Facility Ltd; Melbourne, Australia;
http://www.agrf.org.au/docstore/seq/How_to_Prepare_Your_Capillary_Separation_(CS
)_Samples.pdf). The reactions typically included 400-500 ng of plasmid DNA or 40-50
ng PCR products as templates, 3.2 pmole primer, 0.5 µL BigDye v3.1 and 3.5 µL of
supplied 5X dilution buffer, in a volume of 20 µL. Cycling involved initial denaturation
at 94oC for 5 mins, then 30 cycles of denaturation (94oC, 10 seconds), annealing
(50oC, 5 seconds) and primer extension/termination (60oC, 4 mins). The sample was
mixed with 75 µL 0.2 mM MgSO4 for 15 mins, centrifuged for 15 mins at 14.5 x 1000
g, air-dried, then sent for capillary separation at AGRF using a 3730xl DNA Analyser
(Applied Biosystems; USA).
2.11.2 DNA sequence analysis
The Bioedit sequence alignment editor
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html) v7.0.5 was used for analysis of raw
sequence data and developing contigs from overlapping sequences. Sequence
alignments were performed using the ‘ClustalW Multiple Alignment’ function in
Bioedit for obtaining identity scores, and alignments of CypB genomic with cDNA
sequences were conducted to elucidate introns and exons. The alignments were
displayed using ‘plot identities to first sequence with a dot’ in Bioedit, calculation of
level of similarity was conducted by pairwise alignments, and restriction maps predicted
by the ‘Restriction Map’ option. Coding sequences of CypB genes, deduced from exon
contigs of genomic copies and cDNAs, were translated into putative proteins using the
‘translate or reverse-translate’ option, the amino acid sequences aligned and compared
as above, and the sizes of predicted proteins determined using the ‘Amino Acid
Composition’ option.
54
2.12 Amplification of promoter sequences by inverse PCR (IPCR)
2.12.1 Promoter sequences amplification
The promoter sequences of wheat CypB gene copies were amplified by IPCR as per
Digeon et al. (1999). Based on sequencing results (described in Chapter 3), the enzyme
SacI was chosen for gDNA digestion, as it had no site in the T. urartu CypB gene
(TuCypB-A) and one site within intronV of the Ae. tauschii gene (AetCypB-D).
Genomic DNA (1µg) from T. urartu and Aet. Tauschii was digested with 1µL SacI (10
u/µL) in a volume of 20 µL for 2 hours, phenol/chloroform-extracted, resuspended (at 2
µg/mL) in 500 µL ligation buffer (50 mM Tris–HCl pH 7.4,10 mM MgCl2, 10 mM
dithiothreitol, 1 mM ATP) with 1µL (3 u/µL) T4 DNA ligase and ligated for 15 hours at
15oC. The digestion was purified with a double volume of 100% chilled ethanol and
dissolved in 20 µL dH2O. Using the PCR protocol (section 2.7.2), the first round
(IPCR) was conducted with primer pair CYPBIPCRF4/CYPBF3 and the second round
(nested) PCR was conducted with CYPBIPCR-R1/CYPBF3 (Table 2.7).
2.12.2 Characterisation of amplified fragments
The second round PCR products were cloned and plasmid DNAs purified as above, and
inserts were amplified with CYPBF3/CYPBIPCR-R1 from individual clones. The
products were digested with SacI to estimate the lengths of DNA likely from upstream
and downstream of CypB genes. The clones of interest were partially or fully
sequenced.
2.13 Physical and genetic mapping of wheat CypB genes
2.13.1 Physical mapping of wheat CypB genes
Based on comparison of sequences of CypB genes from common wheat and its
progenitors, the common wheat genes were tentatively assigned to different genomes
(results in chapter 3). Physical mapping onto specific chromosomes involved the use of
amplified fragment length polymorphisms (AFLPs) followed by restriction fragment
length polymorphisms (RFLPs) in the amplified fragments. For AFLP, utilising the
length variations noted in certain sections of CypB genes of common wheat,
amplifications were conducted with the primer pair WC3/CYPBR5 from gDNAs of T.
aestiuvm v. Chinese Spring (CS), the 21 nullisomic/tetrasomic (NT) lines in CS
background (Sears, 1966) (Table 2.5) and 10 deletion lines for chromosome 7 (Endo
55
and Gill, 1996) (Table 2.6). Sequences of the WC3-CYPBR5 sections of CypB genes
from A, B and D genomes of common wheat were used to identify unique restriction
sites by the ‘Restriction Map’ program in Bioedit. PCR products from the NT and
deletion lines with WC3-CYPBR5 were double-digested with HaeIII+NcoI to identify
the presence/absence of specific genes in each line.
2.13.2 Genetic mapping of wheat CypB genes
The genetic mapping was conducted by the following steps.
(i) Design of allele specific primers: AS primers were designed for specific
amplification of CypB genes from the A, B or D genomes in common wheat (results
given in chapter 3). The sequence of AS primers, their positions on genomic sequences
and expected sizes of amplification products are indicated in Table 2.7.
(ii) Confirming specificity of AS primers: partial CypB was amplified with
WC1/CYPBR5 from gDNA of T. asetivum cv. Rosella. Nested PCR was then
performed on these products with AS primers CYPB-A1/CYPBR-A1 (for A genome
allele), CYPBF4/ CYPBR-B1 (B genome allele) and CYPB-D1/CYPBR-D1 (D genome
allele). The second round PCR products were digested with HaeIII for diagnostic
patterns.
(iii) Identification of polymorphisms in parental lines: PCR was performed using
WC1/CYPBR5 on gDNA from single plants of the five pairs of parental lines
(Cranbrook x Halberd; Tasman x Sunco; CD87 x Katepwa; Janz x Kukri; Egret x
Sunstar) (Table 2.7), followed by nested PCR with AS primers. The PCR products
from different genomes in the parental lines were sequenced partially in order to
confirm the noted AFLP or the single nucleotide polymorphisms (SNPs) at the sites of
the restriction enzyme HpyCH4IV, leading to RFLPs (results in chapter 4). Any
polymorphisms between the corresponding genomes in parental pairs were confirmed
directly by analysis of their respective DNAs by AFLP or RFLP.
(iv) Application of the AFLP or RFLP tests to the doubled haploid (DH) progeny lines
from the respective cross, exactly as for parental lines: CypB marker from A genome
was analysed from 161 DH lines generated from the Tasman x Sunco cross by AFLP
and CypB marker from B genome was analysed in 162 DH lines from the same cross by
RFLP.
56
(v) Integration of CypB markers in the linkage map from Sunco x Tasman (Chalmers et
al., 2001) based on the bin mapped markers (Section 4.3.1) and data analysis by Map
Manager software package QTXb20.
2.14 RNA extractions and cDNA synthesis
2.14.1 Tissue samples
Seeds of T. aestivum cv Cranbrook were grown under controlled conditions (described
in section 2.5) for two weeks. The leaves and stems were collected separately from
young seedlings. Developing caryopses were collected 6, 10, 14, 18, 22, 30, and 34
days after anthesis (DAA). All tissue samples were snap-frozen in liquid nitrogen and
stored at -80oC.
2.14.2 Total RNA isolation
Total RNA was isolated with the TRIsure reagent (Bioline) according to the provided
protocol. The samples of plant materials (50 mg) were ground. TRIsure (1 mL) was
added to each tissue and incubated for 5 mins. Chloroform: isoamyl-alcohol (24:1, 0.2
mL) was added, incubated for 2-3 mins, and centrifuged for 15 mins at 14.5 x 1000 rpm
at 2-8oC. The supernatant containing RNA was carefully transferred to another tube,
mixed with 0.5 mL isopropanol, incubated for 10 mins, centrifuged for 10 mins at 2-
8oC. The RNA pellet was washed with 70% ethanol, air-dried, dissolved in 30 µL
DEPC treated dH2O, and stored at -80oC. The RNA concentration was estimated by
spectrophotometry.
2.14.3 First-strand cDNA synthesis
The reaction 1 included RNA (1 µg), 0.5 µg/µL Oligo (dT)18 (1 µL), 10 mM dNTP mix
(1 µL) in a volume of 10 µL made up with the DEPC treated dH2O, and was incubated
for 10 mins at 65oC. Reaction 2 included 5 x RT Buffer (4 µL), RNase inhibitor (1 µL),
and 200 U/µL reverse transcriptase (0.25 µL) in a final volume of 10 µL with DEPC-
treated dH2O. This was added to the reaction 1, incubated for 60 mins at 37oC, and
terminated by incubating for 15 mins at 70oC, then stored at -20oC.
57
2.15 Preliminary analysis of some Cyps and PDILs genes expression
PCR was conducted using 0.5 µL first-strand cDNA (out of 20 µL synthesised above),
0.5 µL of each primer (0.1 µg /µL) and 12.5 µL of 2X BioMix in 25 µL volumes made
up with dH2O. The PCR conditions were initial denaturation of template DNA at 95oC
for 5 mins, followed by 30 cycles of denaturation (94oC, 45 seconds), primers
annealing (at appropriate temperatures, for 45 seconds; Table 2.8), extension (72oC, 1
min), then a final extension (72oC, 5 mins). Simultaneous amplifications of all cDNAs
with actin gene primers designed based on actin cDNA of Triticum aestivum (Genbank
accession no. AB181991) provided the house-keeping gene expression controls. The
PCR products were separated on 1% agarose gels.
Table 2.8 Primers used for gene expression study*
Names of Primer Sequence Expected size (Annealing Temperature)
TaCYP23-1 WC4 5’CGCACAAGGTCTACTTCGA3’ 496 bp (54
oC)
CYPBR1 5’CTGTCGGCAATGACAACC3’
TaCYP24-1
CYP21-1F2 5’GGCCGTCGGGTTCCT3’ 542 bp (52oC)
CYP21-1R 5’CCCCTTCAATGGCGT3’ TaCYP26-2
CYP23-1F2 5’ACGCCTCGGATCCCAACC3’ 447 bp (63oC)
CYP23-1R2 5’GGGTTGGCAGGCGCTCT3’
TaPDIL1-1
PDI-A3F 5’CAACCATCCTTACCTCTTGAAATA3’ 627 bp (62 oC) PDI-A4R 5’ATACGAGACCTTCTTCCCGCTA3’
TaPDIL1-2
PDI-B2F 5’CTGCTTTGGAGAAATTCATTGAG3’ 700 bp (62 oC) PDI-B3R 5’CCCTCGTAGGAGACCTTCTTT3’ TaPDIL1-3
PDI-D5F 5’AAAGAGGATCAGGCACCACTG3’ 727 bp (62 oC) PDI-D6R 5’TACAGTATTTCTCGCAACGGGA3’
ACTIN ACTIN-F 5’GTTTCCTGGAATTGCTGATCGCAT3’ 410 bp (62
oC)
ACTIN-R 5’CATTATTTCATACAGCAGGCAAGC3’
*Primers used for amplification of Cyp genes (TaCYP24-1 and TaCYP26-2) were designed based on the identified TA sequences. Primers used for amplification of TaCYP23-1 were designed based on CypB genomic sequences chapter 3). Expected sizes of Cyp genes were based on the cDNA (chapter 3) or identified TAs. Primers used for amplification of PDIL genes (TaPDIL1-1, TaPDIL1-2, and TaPDIL1-3) were as reported (Johnson and Bhave, 2004a). Expected sizes from first strand cDNA were based on reported sequence data in Genbank (AF262979, AF262980, and AF262981). Primers used for amplification of ACTIN gene were designed based on the cDNA sequence of the actin gene of wheat (Accession No. AB181991).
58
Bioinformatics Methods
2.16 Identification of CypB gene and its promoter sequence in rice
The bacterial artificial chromosome (BAC) clone containing the putative CypB gene of
rice was identified by a BLASTn search of the TIGR (The Institute for Genomic
Research) Rice Genome Annotation Database Assembly 2006
(http://www.tigr.org/tdb/e2k1/osa1/, accessed 11/2006) using TC264488 as the query
sequence. The structure of rice CypB gene was then analysed by alignment with the
experimentally isolated CypB genes of common wheat. The ‘Rice Genome Browser’
interface of the TIGR database was used to find the region upstream of ATG and
analyse it for the putative promoter sequences.
2.17 Identification of putative wheat CypB orthologues at rice CypB
locus
The ‘Rice Genome Browser’ interface of the TIGR Rice Genome Annotation
(http://www.tigr.org/tigr-scripts/osa1_web/gbrowse/rice/; accessed 03/2008) was used
to search for putative wheat orthologues at the rice CypB locus. The search was
involved a few steps: (i) searching the rice genome by entering ‘rice CypB gene BAC
locus (LOC_Os06g49480) (see chapter 3) in ‘landmark or region’; (ii) identification of
‘wheat bin mapped markers’ putatively located at/near the CypB gene of rice by using
‘Wheat Bin Mapped Markers’ feature track; (iii) expressed sequence tags (ESTs) probes
that had been physically mapped to a wheat chromosomal ‘bin’ by searching for probes
used in wheat physical mapping experiments available from the wEST database
(http://wheat.pw.usda.gov/wEST/, accessed 03/2008); (iv) the ESTs sequences were
searched from EST database (http://www.ncbi.nlm.nih.gov/Database/, accessed
03/2008) and aligned with exon contig of the isolated CypB genomic gene (TaCypB-B)
in T. aestivum cv. Rosella (see chapter 3).
2.18 Identification of putative QTLs in rice and wheat at the CypB
locus
The Gramene Genome Browser (http://gramene.org) was used to look up in Oryza
sativa ssp. japonica genome database. The chromosome location of rice (in bp)
containing the putative rice CypB locus (see results) and the list of rice QTL accession
IDs were shown at the Oryza sativa ssp. japonica ContigView (Gramene release V27
59
database) by graphic view. Further, the QTL category, trait name, genetic position and
map set with list of associated flanking markers for each listed QTL were identified by
browsing its ID in the Gramene QTL database. The rice genomic sequence assembly
positions of the flanking markers were searched in Gramene Markers database. In
addition, wheat QTLs located on chromosome 7 long arm (based on results of physical
location of our isolated CypB gene) were identified by searching the Integrated Wheat
Science database (http://www.shigen.nig.ac.jp).
2.19 Rice Cyp and PDIL family identification and analysis
The bacterial artificial chromosomes (BACs) containing the putative Cyp or PDI family
genes in rice (Oryza sativa ssp japonica cv. Nipponbare) were searched in the Rice
Annotation Release 5 (and in Release 6) (http://rice.plantbiology.msu.edu/osa1.shtml,
last accessed 02/2009) using the reported Arabidopsis genomic sequences of loci
encoding Cyps (Romano et al., 2004a) or PDILs (Houston et al., 2005) as queries for
BLASTn searches (searching a nucleotide database using a nucleotide query). The
genomic and coding sequence (CDS; exon contigs) from each representative locus were
used for gene structure analysis. The gene structures were generated by comparison of
the genomic sequences and CDS respectively with the GSDS (Gene Structure Display
Server) program (http://gsds.cbi.pku.edu.cn). Reported cDNA encoding rice PDILs
(Houston et al., 2005) or longest EST contributing to corresponding TA encoding
PDILs or Cyps, respectively, was used as query to generate a map of chromosomal
locations from the Oryza sativa genome view in the NCBI Map viewer
(http://www.ncbi.nlm.nih.gov, last accessed 02/2009). The sequences of CDS were
used as queries to search the TIGR Plant Transcription Assembles (TAs) database on
the website (http://plantta.jcvi.org, last accessed 02/09) by BLASTn for TAs having
100% identity with queries on the comparable parts. The deduced amino acid
sequences of Cyps or PDILs in rice from the exon contigs were downloaded from same
database. All putative proteins of Cyps or PDILs in rice were used for generating the
phylogenetic tree and other comparative biochemical analyses.
60
2.20 Wheat Cyp and PDI family identification and analysis
Transcript Assemblies (TA) encoding putative PDILs or Cyps in wheat (Triticum
aestivum L.) were searched using the same queries as in section 2.19 for BLASTn
searches of TIGR Plant Transcript Assemblies database (http://plantta.jcvi.org, last
accessed 02/2009). The obtained TAs were translated into amino acid sequences by
Bioedit sequence alignment editor v 7.0.5
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html) with the selected reading frame, to
identify whether they were the expected TAs. The deduced amino acid sequences were
used for generating phylogenetic trees and other comparative biochemical analyses.
The chromosomal map positions of the identified wheat PDIL or Cyp genes were
generated progressively (Fig 2.1), i.e. by (i) a search for the putative wheat orthologues
at the rice locus was conducted in the ‘Rice Genome Browser’ interface of the TIGR
Rice Genome Annotation (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/; last
accessed 02/2009) by entering each identified rice PDIL locus or Cyp locus in the
‘landmark or region’ to identify the corresponding wheat chromosomal ‘bin’ mapped
markers; (ii) cDNA probes used for the physical mapping data available in the
GrainGenes structured query language (SQL) database were searched by entering the
name of queries into the ‘Mapped Loci for EST-derived Probes’
(http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi, last accessed 02/2009); (iii)
sequences of 5’ probes were searched for in the EST database
(http://www.ncbi.nlm.nih.gov, last accessed 02/2009); (iv) sequences of probes were
aligned with corresponding wheat TAs for identity calculation; (v) the map for
chromosomal locations was generated based on deletion breakpoints from wheat genetic
and genomic resource centre (http://www.k-state.edu/wgrc/Germplasm/Deletions, last
accessed 06/2009).
61
5’ probe: BE444859
3’ probe: BQ161460
http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi
EST sequence: BE444859
http://www.ncbi.nlm.nih.gov
Southern blotting image
http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi
TaPDIL2-1
99.3%
TaPDIL2-2
98.7%
TaPDIL2-3
86.7%
http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/
1DS : C-1DS3-0.48; 1BS : 1BS10-0.50-0.84
Fig 2.1 Representative chart for identification of PDILs or Cyps chromosomal locations in wheat. Identities are the EST sequence (BE444859) with TAs encoding TaPDIL21-, TaPDIL2-2, and TaPDIL2-3 on the overlapping parts, respectively.
2.21 Identification of putative QTLs related to quality in rice and
wheat at loci encoding PDILs
The Gramene Genome Browser (http://gramene.org) was used to look up in Oryza
sativa ssp. japonica genome the chromosome location of rice (in bp) which contained
the putative rice PDIL loci (chapter 6) and list the rice QTL accession IDs shown at the
Oryza sativa ssp. japonica ContigView (Gramene release V27 database) by graphic
view. Further, the QTL category, trait name, genetic position and map set with list of
associated flanking markers for each listed QTL related to quality were identified by
browsing its ID in the Gramene QTL database. The rice genomic sequence assembly
positions of the flanking markers were searched in Gramene Markers database. In
addition, wheat QTLs related to quality locating on chromosomes containing the PDIL
genes (chapter 6) were identified by searching the Integrated Wheat Science database
(http://www.shigen.nig.ac.jp).
62
2.22 Multiple sequences alignments, cellular location predictions and
phylogenetic analyses
The amino acid sequences deduced from exon contigs (CDS) of the putative genomic
genes in rice and TAs in wheat were aligned with those in Arabidopsis and/or
orthologues in human, using ‘ClustalW Multiple Alignment’ application in Bioedit
(section 2.11.2). The phylogenetic trees of Cyps or PDILs were generated based on
these alignments, using Neighbor-Joining program in MEGA 4.0
(http://www.megasoftware.net). Cellular locations were predicted by the ‘WolF
PSORT Prediction’ (http://psort.ims.u-tokyo.ac.jp) and the TargetP 1.1 Server
(http://www.cbs.dtu.dk/services/TargetP), as well as alignments with respective
orthologues in Arabidopsis. Any signal peptides on proteins were predicted by the
SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP) and chloroplast transit
peptides by the ChloroP 1.1 Server (http://www.cbs.dtu.dk/services/ChloroP).
2.23 Prediction of PDIL expression in different tissues in Arabidopsis
The extent of expression of PDIL genes in different tissues in Arabidopsis was
investigated using the BAR (Botany Array Resource) Arabidopsis eFP (Electronic
Fluorescent Pictograph) browser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi,
accessed 12/2008) (Winter et al., 2007). The AGI ID of each locus encoding PDILs in
Arabidopsis was entered in the Arabidopsis eFP browser to search the expression value
in different tissues and developing seeds. The expression data were transferred to Excel
to produce a figure showing absolute expression.
63
Chapter 3
MOLECULAR CHARACTERISATION OF
CYCLOPHILIN B GENES IN WHEAT AND RICE
64
3 Molecular characterisations of cyclophilin B genes in wheat
species and rice
3.1 Abstract
Cyclophilin B, belonging to the peptidyl prolyl isomerase (PPIase) group of enzymes, is
suggested to be involved in storage protein folding and deposition in the developing
wheat grain. The seed storage protein composition, quantity, assortment, resolution of
aggregates, and deposition into protein bodies via two different routes are all key
phenomena affecting cereal grain quality. The genetic basis of the qualitative
differences in protein subunits is now well understood; however, an understanding of
the genetic basis of the assortment and deposition processes is limited in cereals
including wheat. In this work, genes encoding a potentially important ‘foldase’
enzyme, also a possible chaperone, were identified. The three genes (EU627095,
EU627096, EU627097) represent a putative ER-localised form of cyclophilins, CypB
which is of significance due to the storage protein folding, resolution of aggregates and
assortment processes that occur in the ER of endosperm cells. The genome assignments
of the three genes were conducted by comparison with corresponding genes
(EU627098, EU627099) isolated from the tetraploid progenitor, T. turgidum v. durum,
and one each from the diploid progenitors, T. urartu (EU868840) and Ae. tauschii
(EU627100). The gene structures exhibit seven exons and six introns, the exons being
significantly more conserved than introns. The locus LOC_Os06g49480 encoding the
putative rice CypB gene was also identified from the Rice Genome Annotation
Database Assembly. The putative CypB proteins in wheat and rice are 83% identical
and contain all functional domains of a PPIase and an atypical endosperm
retention/retrieval signal. The promoters isolated by inverse-PCR (T. urartu, Genbank
FJ799756; Ae. tauschii, Genbank FJ799757) and identified from rice genome show a
number of potential tissue specific regulatory elements. The data provide essential tools
for physical and genetic mapping of the gene to assess its association with any quality
traits, as well as identification of genetic variations in this potential regulator of seed
storage protein quality.
65
3.2 Introduction
As introduced in Chapter 1 (sections 1.2, 1.3, 1.4), the wheat endosperm storage
proteins are commonly grouped into the monomeric gliadins and polymeric glutenins,
the latter comprised of low and high molecular weight subunits. The gliadins are
stablised by intra-molecular disulphide bonds and non-covalent interactions that
contribute to dough viscosity, and glutenins largely by inter-molecular disulphide
bonds, the number and distribution of which determine dough elasticity and strength
(reviewed in Gianibelli et al., 2001; Shewry et al., 2002; Shewry and Halford, 2002).
The storage protein bonding, assortment and deposition processes in the wheat
endosperm and the highly structured nature of the gluten polymer imply significant
roles for ER-localised molecular chaperones and ‘foldase’ enzymes. An important
candidate for direct, specific roles in these processes is the enzyme group peptidyl
prolyl cis-trans isomerase (PPIase), with the key function to catalyse the cis-trans
isomerisation of the peptide bond preceding proline residues, a rate-limiting step in the
folding of newly synthesised proteins (Galat, 1993). The Cyps, a class of PPIases,
exhibit highly conserved secondary structures but localise to several cellular
compartments. Plant ER Cyps are functionally versatile, and in addition to PPIase
activity, their other roles - such as protein-protein interactions and chaperone roles
under internal ER stress or environmental stress - may be relevant to storage protein
processes. In support of this, high levels of PPIase transcripts were reported in the
developing grain, coinciding with high levels of expression of storage proteins
(Grimwade, et al., 1996). However, except for the preliminary reports of ER-localised
PPIase in maize (Sheldon, et al., 1996) and high levels of transcripts of PPIases (of
unidentified type) in the developing wheat endosperm (Grimwade, et al., 1996), little
other work has been reported on these genes/enzymes. Previous work in M. Bhave’s
group (Johnson et al., 2004b) led to characterisation of genes encoding three
homologues of cytoplasmic CypAs in wheat; however, direct roles for these in the ER
lumen are unlikely duo to their cytoplasmic location. The current project extends the
work on wheat Cyps. This chapter focuses on isolation and characterisation of genes
encoding potential ER lumen located CypB proteins in common wheat and its
progenitors, their putative promoters, and identification of rice CypB gene and its
promoter from the rice genome database. The results of this work have been published
(Wu et al., 2009).
66
3.3 Results
3.3.1 Amplification and identification of CypB genes from genomic DNA and first-
strand cDNA in T. aestivum
PCR amplification of genomic copies of CypB genes from T. aestivum cv. Rosella was
performed with primer pair WC1-CYPBR5 as described (section 2.7), the putative start
codon being included in the WC1 sequence during primer design (Table 3.1; Fig 3.1).
Table 3.1 Primer pairs used for PCR or IPCR amplification/or sequencing of CypB gene and its promoter*
* This table is part of Table 2.7, reproduced here for convenience.
WC1(ST:126)
CYPBIPCR-F4
WC4 CYPBF3
CYPBR5 CYPBR1 CYPBR9CYPBIPCR-R1
SP(767bp)
Fig 3.1 Position of primers used for PCR or IPCR amplification. The putative TaCypB cDNA from TIGR wheat genome database (http://www.tigr.org/tdb/e2k1/tae1/, Johnson and Bhave, 2004; current accession number TA60203_4565) used for designing primers for amplification and/or sequencing of CypB genes. Arrows pointing down indicate positions of forward primer and arrows pointing up indicate reverse primer. Positions of start (ST, 126bp) and stop (SP, 767bp) codons are indicated.
The amplification led to a product of approximately 1.8 kilo base pairs (kb) size (Fig
3.2A), much larger than the corresponding region of TC264488 (317 base pairs; bp),
suggesting the presence of introns. TC84720, which was reported to encode an ER-
localised CypB (Johnson and Bhave, 2004b), was later found split into TC264493 and
TC264488 on the TIGR website during the current project (current accession number
Forward primer Reverse primer ATc EL* Purpose
WC1 (114-131bp)a
5’ATACGATCCAAGATGGCG3’ CYPBR5 (411-430 bp)
a
5’CTGTGGAACGAACTCCCCTT3’
57
oC
317bp
Fragment (WC1-CYPBR5)
WC4 (253-271bp)a
5’CGCACAAGGTCTACTTCGA3’ CYPBR1 (731-748 bp)
a
5’CTGTCGGCAATGACAACC3’ 54
oC ~1800bp
Fragment (WC4-CYPBR1)
WC1 (114-131bp)a
5’ATACGATCCAAGATGGCG3’ CYPBR9 (943-960 bp)
a
5’ ACGGTGGTGTGTGATGCC3’ 57
oC ~2300bp
Fragment (WC1-CYPBR9)
WC1 (114-131bp)a
5’ATACGATCCAAGATGGCG3’ CYPBR9 (943-960 bp)
a
5’ACGGTGGTGTGTGATGCC3’ 57
oC ~850bp cDNA Fragment
CYPBF3 (366-386bp)a
5’ACAGGTGAGAAAGGCATGGGC
CYPBIPCR-F4 (84-98)b
5’AGGGTGGCGGCGACG3’ 62
oC N/A
IPCR CYPBF3 (366-386bp)
a
5’ACAGGTGAGAAAGGCATGGGC
CYPBIPCR-R1 (126-143 bp)a
5’CCACGCCCTCATCGCCAT3’ 62
oC N/A
T7 5’GTAATACGACTCACTATAGGGC
SP6 5’TATTTAGGTGACACTATAGAAT3’
45oC Clone-dependent
WC3 (299-316bp)a
5’TCGGGTTGTCATGGGACT3’
CYPBR4 (255-276 bp)a
5’CGATGTCGAAGTAGACCTTGTG
Internal sequencing for CypB gene
CYPB IPCR-F2 5’GCGTAGTAGATCAGTAGTAGC3
CYPB IPCR-R2 5’GTGTCGTGGTATTGAA 3’
Internal sequencing for CypB promoter
67
TA60203_4565). In a preliminary attempt to identify inter-genome variation, six clones
(TaA1-TaA6) of this gene section (WC1-CYPBR5) in pGEM-T Easy plasmids were
selected randomly. Their inserts were amplified with the vector-based primers T7 and
SP6, and RFLP analysis performed on amplified inserts with HaeIII (selected due to
being the only enzyme with appropriately spaced sites in the TC264488 sequence for
analysis on agarose gels). The results showed three patterns, the four clones (TaA1,
TaA2, TaA3, TaA6) each had three bands (~1400bp, ~400bp, ~50bp); one clone (TaA4)
had four bands (~650bp, ~500bp, ~200bp, ~50bp) and another (TaA5) has three bands
(~1300bp, ~400bp, ~50bp) (Fig 3.2B). One representative clone of each (TaA1, TaA4,
TaA5) was sequenced (Table 3.2).
In an attempt to get longer gene sections, an overlapping section WC4-CYPBR1 was
amplified from Rosella and led to products of ~2.0kb (Fig 3.2C). Inserts of ten plasmid
clones of this fragment (TaB1-TaB10) were generated with T7/SP6. RFLP analysis of
inserts by HaeIII showed that six clones (TaB5-TaB10) corresponded to the TaA1 type;
three (TaB2, TaB3, TaB4) to the TaA4 type; and TaB1 had a new pattern (Fig 3.2D).
The representative clones (TaB1, TaB2, TaB6) were also sequenced (Table 3.2).
Once the sequences of these six clones provided clues to gene structures, primer
CYPBR9 located at the 3’end of the gene, 196 bp after the stop codon, was used with
WC1 (114-960 bp section on TC264488) for amplification of a longer section from
Rosella. The product was ~2600bp (Fig 3.2E). The inserts of ten randomly selected
clones were analysed by RFLP (HaeIII), but all showed an identical type (Fig 3.2F).
The representative clone (TaC1) was also sequenced (Table 3.2).
Amplification with WC1-CYPBR9 from first-strand cDNA of leaves from T. aestivum
cv. Cranbrook led to a product of ~850bp (Fig 3.2G). Three randomly selected clones
(TaCD1, TaCD2, TaCD4) of this cDNA gene section were also sequenced (Table 3.2).
68
A B C
D E F G Fig 3.2 CypB genomic gene sections and the RFLP polymorphisms (HaeIII) of clones in T. aestivum, cv. Rosella and CypB cDNA in T. aestivum, cv. Cranbrook A: amplification of gene section WC1-CYPBR5; B: polymorphisms of clones TaA1-TaA6; C: amplification of gene section WC4-CYPBR1; D: polymorphisms of clones (TaB1-TaB10); E: amplification of gene section WC1-CYPBR9; F: polymorphisms of clones (TaC1-TaC10); G: CypB cDNA section (WC1-CYPBR9) from Cranbrook.
3.3.2 Sequence alignment of clones from genomic DNA and cDNA in T. aestivum
The sequences of the seven genomic clones were analysed with Bioedit
(http://www.mbio.ncsu.edu/BioEdit/bioedit.html) v7.0.5 (Fig. 3.3; Appendix IIA). The
inserts of TaA1, TaA4, and TaA5 were 1869 bp, 1786 bp, or 1820 bp long,
respectively,. those of TaB1 and TaB6 were 2005 bp; and TaB2 was 1936 bp. TaB6
had two single nucleotide polymorphisms (SNPs) compared with TaB1, one of these
being at a HaeIII site, explaining the extra site in TaB1; this could be a Taq-polymerase
introduced artifact in TaB1. In the overlapping sections, TaA1 had 100% identity to
69
TaB6 and TaA4 had 100% identity to TaB2. Two contigs could thus be generated,
TaA1-TaB6 (2386 bp) and TaA4-TaB2 (2307 bp). The insert of TaC1 was 2606 bp and
100% identical to contig TaA1-TaB6 in the overlapping area. Three copies of CypB in
T. aestivum were thus identified:
(i) clone TaC1 (Genbank accession number EU627095);
(ii) contig TaA4-TaB2 (Genbank EU627096);
(iii) clone TaA5 (Genbank EU627097).
The cDNA clones of section WC1-CYPBR9 of Cranbrook, TaCD1, TaCD2 and
TaCD4, were 846-851 bp (discussed later). The sequence of TaCD4 was compared
with above genomic copies to generate gene structures (Fig 3.3).
Table 3.2 Summary of sequenced CypB gene sections in wheat species
Name (Genbank No) Length Wheat species Resource
TaA1 1869 bp T. aestivum cv Rosella Clones of PCR products from gDNA with WC1-CYPBR5 TaA4 1786 bp
TaA5 (TaCypB-D, EU627099) 1820 bp
TaB1 2005 bp T. aestivum cv Rosella Clones of PCR products from gDNA with WC4-CYPBR1 TaB2 1936 bp
TaB6 2005 bp
TaA4-TaB2 (TaCypB-A, EU627096)
2307 bp T. aestivum cv Rosella
TaC1 (TaCypB-B, EU627095) 2606 bp T. aestivum cv Rosella Clones of PCR products from gDNA with WC1-CYPBR9
TaCD1 846 bp T. aestivum, cv. Cranbrook
Clones of PCR product from cDNA with WC1-CYPBR9 TaCD2 846 bp
TaCD4 851 bp TdA1 1786 bp T. turgidum ssp. durum Clones of PCR products from
gDNA with WC1-CYPBR5 TdA2 1872 bp
TdB1 2008 bp T. turgidum ssp. durum Clones of PCR products from gDNA with WC4-CYPBR1
TdB3 1936 bp
TdA1-TdB3 (TdCypB-A, EU627098)
2307 bp T. turgidum ssp. durum
TdA2-TdB1 (TdCypB-B, EU627099)
2389 bp T. turgidum ssp. durum
TuA 1786 bp T. urartu Clones of PCR product from gDNA with WC1-CYPBR5
TuD2 1629 bp T. urartu Clones of PCR product from SacI digested and circularised gDNA with CYPBF3/CYPBIPCR-R1
TuA-TuD2 (TuCypB-A, EU868840)
3350 bp T. urartu
Aet1 (AetCypB-D, EU627100) 2560 bp Ae. tauschii Clones of PCR product from gDNA with WC1-CYPBR9
70
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ATACGATCCGAGATGGCGATGAGGGCGTGGAGGAGGAGCGCGGCGGCGAGGGCCCCGGCGCACCTGTGCCTGTGGCTGGCGCTCGTCGCCGCCACCCTGGTGCTCGCCCAGGTGCTCATCCACCTCGTCCGTCTTCCGCGAACCCATCCC
TaCypB7A ......................................................................................................................................C..........C...T
TaCypB7D .................................C....................................................................................................A...............
TdCypB7B ......................................................................................................................................................
TdCypB7A ......................................................................................................................................C..........C...T
TuCypB7A ......................................................................................................................................C..........C...T
AetCypB7D .................................C....................................................................................................A...............
TuCYPB-A ......................................................................................................................................C..........C...T
Tac4 ...............................................................................................................---------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B CTTACTTTTCCGCTTGCCGATTTGTTCGCCGATCTGATCGTTTCTCTGATACGGTTAATCGTTGCCCTCGCGCTGATGTCTGAACCTGTCTGCCGCGGATCCGGATGGCTTATATGTCTTCGGCGCTGCTGATTTGTTAGGGTTTGGTGT
TaCypB7A ...TAC.......CGCT.CCCCCTC....TCC..C.TG..CCGA......-..T.CCTCT.C.A.GA.T..T.----.CTGAT.T....T...........G..GC..A...G....G..T..G.T.....G.C............A.--
TaCypB7D .........................................................................................................C...........------------------...........A...
TdCypB7B ......................................................................................................................................................
TdCypB7A ...TAC.......CGCT.CCCCCTC....TCC..C.TG..CCGA......-..T.CCTCT.C.A.GA.T..T.----.CTGAT.T....T...........G..GC..A...G....G..T..G.T.....G.C............A.--
TuCypB7A ...TAC.......CGCT.CCCCCTC....TCC..C.TG..CCGA......-..T.CCTCT.C.A.GA.T..T.----.CTGAT.T....T...........G..GC..A...G....G..T..G.T.....G.C............A.--
AetCypB7D .........................................................................................................C...........------------------...........A...
TuCYPB-A ...TAC.......CGCT.CCCCCTC....TCC..C.TG..CCGA......-..T.CCTCT.C.A.GA.T..T.----.CTGAT.T....T...........G..GC..A...G....G..T..G.T.....G.C............A.--
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GGTTGGTGTCTCTGTATGGGATGCGTACTTACGATTTTCTTCCTTGTTTTCAGGGCAAGAAATCCAACCTGTCTGAGGTGACGCACAAGGTCTACTTCGACATCGAGATCGACGGCAAGCCCGCAGGTCTGTGCTGAACCCCTGGCCACA
TaCypB7A --C........G.......-......G.....A........TG.G....C.....T.....G..G........G.............................T.....................................G....A...
TaCypB7D ......................................................................T............................................................................G..
TdCypB7B ......................................................................................................................................................
TdCypB7A --C........G.......-......G.....A.........G.G....C.....T.....G..G........G.............................T.....................................G....A...
TuCypB7A --C........G.......-......G.....A........TG.G....C.....T.....G..G........G.............................T...................T.................G....A...
AetCypB7D ......................................................................T............................................................................G..
TuCYPB-A --C........G.......-......G.....A........TG.G....C.....T.....G..G........G.............................T...................T.................G....A...
Tac4 -----------------------------------------------------.........................................................................------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B AATTTGTTTTCATATCAGCGGGATGTATCATCCCAGTATGGGGCAGGCAGTAGACTGCTACTAATACAACCGATATTGCATGCCTGATTCCTAGTCCTAGCCTAGAAAGCACGGTTTGTAGTTACACTTCGAGTGATTCCTTTTGATTAG
TaCypB7A G....A......G.......C...........T...........G.A...............G.............C...............G.C...............C..............G.G..........A......C....
TaCypB7D G....A......G.......C...............C.........A...............G........A....C.........T....------.G...........C.............G..G.T...............C..T.
TdCypB7B ......................................................................................................................................................
TdCypB7A G....A......G..T....C...........T...........G.A...............G.............C...............G.C............G..C..............G.G..........A......C....
TuCypB7A GG...A......G.......C...........T...........G.A...............G.............C...............GTC...............C..............G.G..........A......C....
AetCypB7D G....A......G.......C...............C.........A...............G........A....C.........T....------.G...........C.............G..G.T...............C..T.
TuCYPB-A GG...A......G.......C...........T...........G.A...............G.............C...............GTC...............C..............G.G..........A......C....
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
S.T. HaeIII
Exon I Intron I
Exon II Intron II
CYPBIPCR-R1 CYPBIPCR-F4
71
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GATCGTTGAAAAA--TGTGAGATTGACATTGAAATTGAATTTCCATGTTTAGATGGCTGCACTGCACCTAATTTTGTTTTATGTTTA---GTGAGAAAAATATTCACTGGTGTGCTATGCATGTACGTTCTTAATGGTTTAACTCCTTTT
TaCypB7A .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
TaCypB7D .............AT........C..................T...................C..G.....................TAA............................T......T.......T..--.GC.TCT....C
TdCypB7B ......C.....G--........................................................................---............................................................
TdCypB7A .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
TuCypB7A .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
AetCypB7D .............AT........C..................T...................CA.G.....................TAA............................T......T.......T..--.GC.TCT....C
TuCYPB-A .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B CATTGTTTGATTGATCCGCTGGCATTTAAATTGATTCTGAATTGTCATAGGTCGGGTTGTCATGGGACTTTTCGGCAAGGCTGTTCCTAAAACCGCAGGTATCATCTCCTTACTGTTTGAGTTAAAAACCATGTACTCATCTATTGGATG
TaCypB7A ----..............T..........................................................................................C.G.....C................................
TaCypB7D ..................G..................................................................................................C......C...............T..C......
TdCypB7B ...................................................................................................................................C..................
TdCypB7A ----..............T..........................................................................................C.G.....C................................
TuCypB7A ----..............T..........................................................................................C.G.....C................................
AetCypB7D ..................G..................................................................................................C......C...............T..C......
TuCYPB-A ----..............T..........................................................................................C.G.....C................................
Tac4 --------------------------------------------------................................................----------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ACAGAAATGCTAATGGCTTGATATCTTTTGTTCTACAGAGAACTTCAGAGCACTCTGCACAGGTATGCTTCCCTACCACTTGCGCTGTTTTCTATATGTTATAAGCGTTATGCCGTCTGCTTGTTGAAGTTTCTAGATGATTAGTCTGAT
TaCypB7A ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
TaCypB7D ..................C.....................................................G...T............-----------.C.TT.T...C...............G......C................
TdCypB7B ......................................................................................................................................................
TdCypB7A ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
TuCypB7A ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
AetCypB7D ........................................................................G...T............-----------.C.TT.T...C...............G......C................
TuCYPB-A ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
Tac4 --------------------------------------.............G..........----------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GGCAAGGCTGGCTATTGGAATAAAAT-TTGCTCTATACAATCACAGTTGCTTTGTTATTTTGGCTCACATCTTCAAGTAATTTAGCCAATCAAATGTAATTGGCTTCCTCAGAAAGTCAAAACATAATTGTACAGTGGAAATTATATACT
TaCypB7A ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
TaCypB7D ............GT............-C........C....T..T...A...-............A--..........T................A......C...........................C...A...........----
TdCypB7B ..........................-..............................................A............................................................................
TdCypB7A ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
TuCypB7A ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
AetCypB7D ............GT............-C........C....T..T...A...-............A--..........T................A......C...........................C...A...........----
TuCYPB-A ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon III Intron III
Exon IV Intron IV
HaeIII
WC3
72
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GTAACTAATTTGATGCATGCTATGCTTGTAACATGGAAACGTTG--CTTGCCTTTTTCTTATAAAAGAAGGAAGCTATCTGTATTGTTCCCTTGTTATTTCAAATCCAAGTATACCTTTCTAGACACGACA---TAGAGAGAATGGTGGC
TaCypB7A -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
TaCypB7D -.....C.............................C..A.C..TG...A........C.TG..G........T...C..........T...CA.................T....A..........A.G.ATG................
TdCypB7B ............................................--.....................................................................................ACG................
TdCypB7A -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
TuCypB7A -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
AetCypB7D -.....C.............................C..A.C..TG...A........C.CG..G........T...C..........T...CA.................T....A..........A.G.ATG................
TuCYPB-A -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B AGCCTGTTTGCCTGCAAATTTTTGGGCATGATCTGTTTAAATCCCAAGCTCCCTAGAGCATCATTGAAGGGTTCTAAGTTGTATGTTCTACACAGTCTCTAGTAGTAGCCCTGTGTGTGGA-TTTTGATTGTTCATCCCTCCTCTACCAT
TaCypB7A ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
TaCypB7D .........T........G.......G..C............T....A.C....C......A........-.....T......A.....G...G.....A..G..............CA..A..................TG--------
TdCypB7B .........................................................................................................................-............................
TdCypB7A ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
TuCypB7A ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
AetCypB7D .........T........G.......G..C............T....A.C....C......A........-.....T......A.....G...G.....A..G..............CA..A..................TG--------
TuCYPB-A ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GGTTCTAAAAGAAATCCCTCTACCATACAAGTGCCCAAATATGAATGTCAATAAGAAAAAA-GAGAAGAAAT-AGATAACTGGTTGAATTTGTAGTTGGCTGGTGCCTAGCTGAATATATC--AGAGTATTTTACTTCATGGAAA--AAG
TaCypB7A -----------------.................-T.............T...T.......A..........G.AG...........G..C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
TaCypB7D -----------------..................T.................T.......-..........T.AG.......C...G..C.......T..A........-------.C.AA--.TA..............A...GA...
TdCypB7B ..........A........G.........................................-..........-................................................A--.....................--...
TdCypB7A -----------------.................-T.............T...T.......A..........G.AG...........G..C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
TuCypB7A -----------------.................-T.............T...T.......A..........G.AG..............C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
AetCypB7D -----------------..................T.................T.......-..........T.AG.......C...G..C.......T..A........-------.C.AA--.TA..............A...GA...
TuCYPB-A -----------------.................-T.............T...T.......A..........G.AG..............C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GTGGATAAATGTTGTCATACTCTTCATA--------TTCCACCATAGAGTATTACGAAAGCCTATCATTATTTACTGCATATATCATTATATTCAGAATATCTGCTTTATTAGTACTACAGTCTGTATGGTAGCTT--------GATATC
TaCypB7A ........C...C..T.C...--.....--------......................G.............................G....................C..................T.......CTCTCTAT......
TaCypB7D ........C...C..T.C..........AGTTCATA.....A.........................C..............-................................A....A.......A.......CTCTCTAT...G..
TdCypB7B ............................--------....................................................................................................--------......
TdCypB7A ........C...C..T.C...--.....--------......................G.............................G....................C..................T.......CTCTCTAT......
TuCypB7A ........C...C..T.C...--.....--------......................G.............................G....................C.............C....T.......CTCTCTAT......
AetCypB7D ........C...C..T.C..........AGTTCATA.....A.........................C..............-................................A....A.......A.......CTCTCTAT...G..
TuCYPB-A ........C...C..T.C...--.....--------......................G.............................G....................C.............C....T.......CTCTCTAT......
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
NcoI
HaeIII
73
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B TGCCACTGGTTGCTGACCATGCCCCACTTAATTGTCATACAGGTGAGAAAGGCATGGGCAACAGCGGCAAACCTCTCCACTACAAGGGGAGTTCGTTCCACAGAATTATCCCCAGCTTCATGATCCAAGGAGGGGACTTCACTCTTGGCG
TaCypB7A ...T.G.....................................A..........................G...............................................................................
TaCypB7D ...T.G..........................C..........A........T..................................................-----------------------------------------------
TdCypB7B .....G................................................................................................................................................
TdCypB7A ...T.G.....................................A..........................G...............................................................................
TuCypB7A ...T.G.....................................A..........................G................................-----------------------------------------------
AetCypB7D ...T.G..........................C..........A........T................................................................................C..........C...T.
TuCYPB-A ...T.G.....................................A........T.................G..............................................................T................
Tac4 ------------------------------------------............................................................................................................
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ATGGAAGGGGCGGTGAATCAATCTATGGCACAAAGTTCGCCGATGAGAACTTCAAGCTCAAGCACACTGGACCAGGTAATGATCCGTGTTTCTTGAAATCCAGTTC-CAACTGCAAGTGA-AC-GATAGAATGATAGCCGGGGTTAAGAT
TaCypB7A .........................C................................................................................-............CT..-.G.....A.....G.C..........
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ..........................................................................................................-.............-..-..........................
TdCypB7A .........................C................................................................................-............CT..-.G.....A.....G.C..........
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ..........T..............C...........................................................T................T..TG......G...A.CTC.-.T.............CT.......CC
TuCYPB-A .........................C................................................................................-............CT..-.G.....G.....G.C..........
Tac4 ...........................................................................---------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GTCCTCCTTTTTCATTGACTGGTTGTTGGTGCAGGCTACCTTTCCATGGCCAATGCTGGGAGAGATACCAATGGATCCCAGTTCTTCATCACCACTGTAACCACGAGCTGGTAATT-TTCATGA--CTCTGTGATTGCAGCAGTTTGTAC
TaCypB7A ..........................C.........................................................................................-.......TT..T.............T....CG.
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ....................................................................................................................-.......CT..--....................
TdCypB7A ..........................C.........................................................................................-.......TT..T.............T....CG.
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D A.........................C............................................C.............................T..............C....C..TG..T...A..C....T.A.....G.
TuCYPB-A ..........................C.........................................................................................-.......TT..T.......A.....T....CG.
Tac4 ----------------------------------............................................................................----------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B TAAGCCTTT-TATTCTGCTTGAA-------CTTTGTTGTTGATGATCTACATTTTGTATGTTAGGTTGGACGGCAAGCACGTCGTGTTCGGCAAGGTGTTGTCTGGAATGGACGTGGTCTACAAGGTTGAGGCCGAGGGCAAGCAGAACG
TaCypB7A .G.AT....C.G.......G...-------..C.A...........A....C.C.....TC.........T...............................A...............................................
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B .........-.............-------........................................................................................................................
TdCypB7A .G.AT....C.G.......G...-------..C.A...........A....C.C.....TC.........T...............................................................................
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ...A.....-.G.....T.G.G.GTTGGGA................A....C.C.....TC.........T........................................................C......................
TuCYPB-A .G.AT....C.G.......G-------GAA..CCA...........A....C.C.....TC.........T...............................................................................
Tac4 ----------------------------------------------------------------......................................................................................
Exon V
Intron V
SacI
Exon VI Intron VI
Exon VII
HaeIII
HaeIII
CYPBF3 CYPBR5
74
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B GGACGCCAAAGAGCAAGGTTGTCATTGCCGACAGCGGTGAAGTGCCGCTGTGATGAGACCTGAAATGGGCTCTGGTGGAA-GCTACGGTGACTAGTTTTCGTTGTAGAACCGTTACTAGCCGGTCTGTTTTTTGTTTTCGTGTACTTTTG
TaCypB7A ............A.....................--------------------------------------------------------------------------------------------------------------------
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ..................................--------------------------------------------------------------------------------------------------------------------
TdCypB7A ..................................--------------------------------------------------------------------------------------------------------------------
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ....A...........A...........T........G..................--.......C..T...........A.................C..........G.....T..T....A.-------.A.............CC.
TuCYPB-A .........................C...........A..............................T...........A...................A..............T.GT.G..A.-------...............CC.
Tac4 ................................................................................-...................................................................C.
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B TGTGAAATTCAGACGACAAGGATTCTC----TTGCTATAAGAAAGTATAATGACTTGCGGATTTACCCGTGATCTGGCTGGCAACTTTGAAGGCATCACACACCACCGT-----------------------------------------
TaCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ......-..T.....G....TT..T..CTCT..A......A......C..........----...............................T...............-----------------------------------------
TuCYPB-A ......-..T.....G...A.C..T.TCTCT.-A......A..........A......----....T.A....A...A..............TGC..TT.TTATTTTT.CTTGGATGTTTTGCTGGGTTATTTGCCTTGGAGAAAGTGCT
Tac4 ...........................----...........................----C..............................................-----------------------------------------
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCYPB-A GATATGCGCCTTTTGATTTGAACGATTATCGATCGTCAGCAAACAAATATTCTCGCTTAGTACTGCAAGTGCAGCGGCCGATACCTCCCGCCTGGTGATTGTTTTTTTATGAGGACCAATCTCACAGTAGTTTTATACTTGAAAATCTTT
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCYPB-A AAAGAAACATTTCTGGCTTTACGCTTTAAAAGGTCTGAAAACTGTTGGGAGCACTACCCTATGACCTTTGTGTCGATGAAGCTCCACCACTGCCCGGTAGAGGAGAATCTCCAACAAACCTCCACCACCATAGACCACAGTAGTATTCTT
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
S.P.
75
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCYPB-A TTTCCCTGGCAGAAAAAGAAATATTCTTTGTTAAAAAAAGAGAAAGAAATATTCTCTTACAATCCTATTTGTTTTTCTTATACGATAATAATCTACGGTCCGCATCTTCTTCTCATACGATTGGTACTATGAATATACGTAAAAAAAGTA
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCYPB-A AACAGAGTCCGCGGTTTTAAGAAATAGGAAAAGAAAAGGAGTTATGCAAAGACACGGCGAGCAACCCTAGAAGCCGCATCTTCCACTCAACCAATATATTTTTCTTTCATCTAGTATATCATATTAAAAAAAAGGAGGAATCCTACAATC
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7B ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCypB7A ------------------------------------------------------------------------------------------------------------------------------------------------------
AetCypB7D ------------------------------------------------------------------------------------------------------------------------------------------------------
TuCYPB-A AGCATCAGGGCCGCTGGCTCCAGATCGGACGGTGGAGCGGGCAGAGCCGCGCGCCCAGGGGGCCTAATAATTGGCGTCCCCCCATCCGCCCCCGAGCCGCCCCCAAACCCCGTCCCGTCAAACGCAAAGAAGACAAGCGCAGCAAGTAGG
Tac4 ------------------------------------------------------------------------------------------------------------------------------------------------------
3460 3470 3480 3490
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB7B ----------------------------------------
TaCypB7A ----------------------------------------
TaCypB7D ----------------------------------------
TdCypB7B ----------------------------------------
TdCypB7A ----------------------------------------
TuCypB7A ----------------------------------------
AetCypB7D ----------------------------------------
TuCYPB-A GTTAGGAGAGCCGACGGCGGCGGCGGCGGCGGAAAGCGAG
Tac4 ---------------------------------------- Fig 3.3 Alignment of sequence from seven CypB genomic genes in wheat species. Dots indicate bases identical to the top line, broken lines indicate gaps. Start (S.T.) and stop (S.P.) codons, primers used for IPCR and physical mapping are underlined. The digestion sites of HaeIII, NcoI, SacI are framed.
76
3.3.3 Identification of two CypB genes in T. turgidum ssp. durum
In order to identify which copies of genes from T. aestivum cv Rosella were from the A
or B genome, gene sections WC1-CYPBR5 and WC4-CYPBR1 were amplified from
gDNA of T. turgidum ssp. durum, and led to products of sizes similar to those in T.
aestivum (data not shown). Six randomly selected clones of WC1-CYPBR5 showed
two HaeIII RFLP types, type I (clones TdA1, TdA3, TdA5) and type II (clones TdA2,
TdA4, TdA6) (Fig 3.4A). One of each (TdA1 and TdA2) was sequenced and found to
be 1786bp and 1872bp, respectively (Table 3.2; Appendix IIA). Ten randomly selected
clones of WC4-CYPBR1 were similarly analysed and showed two RFLP types (Fig
3.4B). One representative of each (TdB1 and TdB3) was sequenced and found to be
2008bp and 1936bp respectively (Table 3.2; Appendix IIA). Further, TdA1 was 100%
identical to TdB3 and TdA2 was 100% identical to TdB1 in the overlapping areas
(Appendix IIA). Two contigs of CypB genes in T. turgidum ssp. durum were thus
generated (Fig 3. 3):
(i) 2307bp (TdA1-TdB3; Genbank EU627098)
(ii) 2389bp (TdA2-TdB1; Genbank EU627099)
A B C Fig 3.4 RFLP Polymorphisms (HaeIII) of CypB genomic clones in tetraploid and diploid wheat A: Clones (TdA1-TdA6) with insert fragment (WC1-CYPBR5) in T. turgidum ssp. durum; B: Clones (TdB1-TdB10) with insert fragment (WC4-CYPBR1) in T. turgidum ssp. durum; C: Clones (Aet1-Aet6) with insert fragment (WC1-CYPBR9) in Ae. tauschii.
77
3.3.4 Identification of CypB genes in T. urartu and tentative genome assignments
The section WC1-CYPBR5 amplified from gDNA of a single plant of T. urartu
produced the same size band as in Rosella (data not shown). The PCR product (TuA)
was sequenced directly and was 1786bp (Appendix IIA, Table 3.2). In order to isolate
the full length CypB gene in T. urartu, inverse PCR (IPCR) was applied to SacI digested
and circularised gDNA (section 2.12), as SacI has no site in section WC1-CYPBR5
from T. urartu (Appendix IIA). First round PCR (IPCR) was amplified with primer pair
CYPBF3-CYPBIPCR-F4 (data not shown). Second round PCR on these with
CYPBF3-CYPBIPCR-R1 led to a product of ~2200bp (Fig 3.5A). The section
CYPBF3-CYPBIPCR-R1 was cloned and two clones (TuD1 and TuD2) used further.
Amplification of the inserts with CYPBF3-CYPBIPCR-R1 led to only TuD2 producing
the expected size (~2200bp). Its digestion by SacI gave two bands of sizes (~1700bp,
~500bp) (Fig 3.5B) and its sequence was 1629 bp up to the SacI site (G’AGCTC)
(Appendix IIA, Table 3.2). TuD2 was 100% identical to TuA in the overlapping part.
A 3350 bp contig of CypB gene was thus generated, designated TuCypB-A (Genbank
EU868840) (Fig 3.3). It was similar to the contigs TaA4-TaB2 and TdA1-TdB3 in the
comparable parts. TaA4-TaB2 and TdA1-TdB3 were thus deduced to be from the A
genome and renamed as TaCypB-A and TdCypB-A, respectively.
2500bp 1500bp
2027bp
947bp
564bp
1 2
A B
Fig 3.5 Section amplified by inverse PCR in T. urartu (A) and confirmation of clones with expected inserts (B). Section (CYPBIPCR-R1/CYPBF3) was amplified from SacI digested and circularised genomic DNA. Arrow in A indicates the isolated section of CYPBIPCR-R1/CYPBF3. Arrows in B indicate the right sizes of PCR products and digestion by SacI. Lane 1 in B: PCR products by primer pairs CYPBIPCR-R1/CYPBF3; Lane 2 in B: digestion of PCR products by SacI.
78
3.3.5 Identification of CypB genes in Ae. tauschii and tentative genome assignments
Section WC1-CYPBR9 amplified from gDNA of Ae. tauschii also yielded a ~2600bp
product (data not shown). Six randomly selected clones showed a common HaeIII
RFLP pattern (Fig 3.4C). One clone (Aet1) was sequenced, gave a length of 2560 bp
(Appendix IIA, Table 3.2) and found to be similar to TaA5; AeT1 was hence designated
as AetCypB-D (Genbank EU627100) and TaA5 as TaCypB-D (Fig 3.3). Sequences of
TaC1 and TdA2-TdB1 were most similar to each other, and considering the above
information, these were deduced to be from B genome and renamed TaCypB-B and
TdCypB-B, respectively.
3.3.6 Intron/exon structures and sequence identities of various CypB genes
The sequence of the cDNA clone (TaCD4) was compared with that of CypB genomic
contigs/or clone sequences (Table 3.2) for elucidation of the exon/intron structure
(Appendix IIA, Figs 3.3). The CypB genes in T. aestivum cv. Rosella, i.e. TaCypB-B
and TaCypB-A, both contained 7 exons and 6 introns, and TaCypB-D contained 5 exons
and 4 introns. TdCypB-A and TdCypB-B in T. turgidum ssp. durum, TuCypB-A in T.
urartu, and AetCypB-D in Ae. tauschii, all contained 7 exons and 6 introns (Fig 3.6).
Interestingly, the single domain cytoplasmic Cyp genes in plants, e.g., rice Cyp2
(Buchholz et al., 1994), CyPA genes in wheat (Johnson and Bhave, 2004b) and
AtCYP18-3, AtCYP18-4, AtCYP19-1, AtCYP19-2, AtCYP19-3 in A. thaliana
(Romano et al., 2004a), are all intronless. The Cyp genes encoding ER-localised
isoforms (AtCYP19-4 and AtCYP20-1) in A. thaliana (Romano et al., 2004a) and the
CypB genes isolated here all contain introns ranging from 90bp to 851bp. In the
comparable sections, the A genome genes from common wheat, durum and T. urartu
had introns and exons of identical sizes, as did the D genome genes from common
wheat and Ae. tauschii and the B genome genes of common and durum wheat, except
for intron IV in the durum gene with 3 bp-insertion.
Three genomic genes in common wheat showed exons were conserved with no gaps and
a few SNPs in exon II, V and VII (Fig 3.3). In Exon II, TaCypB-A differed from that of
TaCypB-B by five SNPs and of TaCypB-D by these as well as an additional one. In
exon V, TaCypB-A differed from TaCypB-B by two SNPs and TaCypB-D by two others,
while in exon VII, it differed from TaCypB-B by only one SNP, which did not lead to
amino acid changes (introduced later).
79
242 369 90 851 106 93 111 73 48 24 183 76 136 204
SP 7aa R F W H EVPL
232 320 90 827 107 96 111 73 48 24 183 76 120
TaCypB-B
224 366 90 823 111 73 48 24 61
TaCypB-A
TaCypB-D
232 320 90 827 107 96 111 73 48 24 183 76 120
232 320 90 827 107 96 111 73 48 24 183 76
242 369 90 854 106 93 111 73 48 24 183 76 120 TdCypB-B
TdCypB-A
136 1162
224 366 90 823 107 103 111 73 48 24 183 76 136 195
TuCypB-A
AetCypB-D
249 396 93 466 108 99 255 79 48 24 183 76 136 265 Rice
SP 7aa R F W H ELPM Fig 3.6 Gene structures of the CypB genes isolated from wheat species and the CypB gene identified in rice. Boxes indicate exons, solid lines between them indicate introns, and the numbers show their lengths in base pair (bp). Arrows pointing down or up indicate restriction sites for HaeIII and NcoI in the three copies of genes (TaCypB-A, TaCypB-B, TaCypB-D), respectively. Rice CypB gene structure identified from TIGR (http://www.tigr.org/tdb/e2k1/osa1/, last accessed 11/08). The putative N-terminal signal peptide (SP), the seven-amino-acid insertion characteristic plant Cyps, three essential residues (R, F and H) for PPIase catalytic activity, conserved W for CsA binding, and putative unusual ER-localisation signal sequence EVPL in TaCypB-B or ELPM in rice CypB at the C-terminal are also indicated.
The major variations were in introns I, II and IV. Intron I of TaCypB-B (242bp) was
10bp or 18bp longer than in TaCypB-A or TaCypB-D respectively; intron II (369bp) of
TaCypB-B was 49bp or 3bp longer than TaCypB-A or TaCypB-D respectively, and
intron IV of TaCypB-B (851bp) was 24bp or 28bp longer than TaCypB-A or TaCypB-D.
Intron III was the least variable, with identical lengths but some SNPs. Variation in
introns of the three common wheat genes resulted in certain diagnostic restriction sites
(Figs 3.2, 3.3, 3.6).
Comparison of genomic copies in wheat species and their homologues in Arabidopsis is
shown in Table 3.3. TaCypB-A had 85.8% and 84.2% genomic sequence identity to the
other two common wheat genes, but its identities to TdCypB-A of durum and TuCypB-A
of T. urartu were >99%. The identity of TaCypB-B to TdCypB-B in durum was 99.3%
and that of TaCypB-D to AetCypB-D in Ae. tauschii was 99.8%. The identities of the
TaCypB-B gene were 54.7% over the full length and 83.3% over exon contig when
80
compared to the putative rice CypB (see below). Comparisons with AtCYP19-4 and
AtCYP20-1 in Arabidopsis (Romano, et al., 2004a) showed TaCypB-B had identities of
31.1% (AtCYP19-4) and 37.2% (AtCYPB20-1) over full gene, and 69% (AtCYP19-4) and
67% (AtCYP20-1) over exon contigs.
Table 3.3 Identities amongst the wheat CypB genes and with rice and Arabidopsis orthologs
Genes Ta-
CypB-A Td-
CypB-A
Tu- CypB-
A
Ta- CypB
-B
Td- CypB-
B
Ta- CypB-
D
Aet- CypB-D
Rice AtCYP-20-1
AtCYP-19-4
AtCYP19-4 f
31.7 a 31.8 a 24.9 a 31.1 a 31.9 a 24.8 a 30.3 a 37. 1a 41.0 a -
67.3 b 67.3b 60.5 b 69.0 b 69.2 b 61.4 b 69.2 b 67.1 b 75.0 b -
72.3c 72.3c 61.4c 75.1c 74.9c 62.4c 74.9c 72.7c 82.4c -
AtCYP20-1 e
41.0 a 41.2a 29.4a 37.2 a 37.5 a 28.6a 37.1 a 41.4a -
65.1 b 65.1 b 57.7 b 67.0 b 70.0 b 57.0 b 70.0 b 67.7 b -
73.0c 73.0c 62.1c 75.8c 75.6c 63.1c 75.6c 73.6c -
Rice d
54.7 a 54.7 a 44.1a 54.7 a 53.8 a 44.7a 54.8a -
82.5b 82.5b 77.4 b 83.3 b 83.4 b 78.0 b 83.4 b -
82.7c 82.7c 72.2 c 83.2 c 83.6 c 73.1 c 83.6 c -
Aet- CypB-D
85.6 a 87.3 a 83.9 a 87.8 a 87.8 a 99.8 a -
96.7 b 96.5 b 96.8b 97.5 b 97.5 b 100b -
99.5c 99.5c 100c 99.5 c 99.5c 100c -
Ta- CypB-D
84.2a 84.0a 84.0 a 87.3 a 87.3 a -
97.2 b 97.2b 96.8b 98.7 b 98.7 b -
100c 100c 99.0c 100c 100c -
Td- CypB-B
85.7 a 85.8 a 83.4 a 99.3 a -
98.3 b 98.3 b 97.5b 100 b -
100c 100c 99.0c 100c -
Ta- CypB-B
85.8 a 85.7 a 83.4a -
98.6 b 98.6 b 97.5b -
100c 100c 99.0c -
Tu- CypB-A
99.7a 99.4 a -
99.7b 99.7b -
99.0c 99.0c -
Td-CypB-A
99.6 a -
100 b -
100c -
Ta- CypB-A
-
-
-
Identities of: agenomic sequences;
bexon contigs;
cputative CypB amino acid sequences
deduced from exon contigs. d
Rice CypB identified from (http://www.tigr.org/tdb/e2k1/osa1/, accessed 03/08);
eAtCYP20-1 and
fAtCYP19-4 reported by Romano et al. (2004a).
Sequences at the exon/intron junctions and putative branchpoint sequences of TaCypB-
B are listed in Table 3.4. All junctions followed the universal GT-AG rule, but some
variation was noticed (Fig 3.3). T was the most abundant nucleotide (40-70%) in six
introns in the area of -5 to -14 bases of the 3’ splice site, considered very important in
branchpoint definition and 3’ splice site identification in introns of vertebrate and yeast
81
(Baynton et al., 1996). The branchpoint motifs (YTNAN) are considered very
important splicing signals in most plant introns (Simpson et al., 1996). All TaCypB-B
introns matched this motif in the region 15 nucleotides (intron 2 and intron 5) to 25
nucleotides (intron 4) upstream of the 3′ splice sites. It is interesting to note that the
intron sequences shown in Table 3.4 are also highly conserved among the other six
wheat CypB genes.
Table 3.4 Sequences of exon/intron junction and putative intron branch-points in TaCypB7B
Exon
junction Nucleotide and deduced amino acid sequences
a
Intron number
Splice site sequencesb
5' splice site -- branch point -- 3' splice site -5/-14t %
1-2 CTC GCC CAG-GGC AAG 1 gtgctc ----------cttac -----7c----- ttccttgttttcag 70
Leu Ala Gln Gly Lys
2-3 CCC GCA G-GT CGG GTT 2 gtctgt ----------.ttgat -----1----- ctgaattgtcatag 40
Pro Ala Gly Arg Val
3-4 ACC GCA G-AG AAC TTC 3 gtatca ----------ctaat -----10----- cttttgttctacag 70
Thr Ala Glu Asn Phe
4-5 TGC ACA G--GT GAG AAA 4 gtatgc---------- ctgac -----11----- taattgtcatacag 50
Cys Thr Gly Glu Lys
5-6 GGA CCA G-GC TAC CTT 5 gtaatg ---------- ttgac -----1----- ggttgttggtgcag 50
Gly Pro Gly Tyr Leu
6-7 ACG AGC TG-G TTG GAC 6 gtaatt ----------- ttgat -----7----- attttgtatgttag 60
Thr Ser Trp Leu Asp
.
Consensusd
: g100t100a67 t50 t50t33...y100
t100n a100n...-5-14tttttttttt a50 c67a100g100 aCodons of some amino acids are split across two exons;
bconserved residues with the plant
consensus sequences shown in bold; cnumber of nucleotides between adjacent sequences;
doccurrence (%) in TaCypB7B gene of the most conserved bases in the consensus sequences
was calculated using the splice site sequence data presented in this table. Percentage of ‘t’
nucleotide occurrence in 3’ splice site in the position -5-14 ranged from 40% to 70%.
3.3.7 Alignment of exon contigs from genomic sequences with cDNA clones
The exons of CypB genes (Fig 3.3) were joined together to create exon contigs for
comparison and translation purposes (Figs 3.8). Of the cDNA clones of section WC1-
CYPBR9 of Cranbrook, TaCD1 and TaCD2 were 846 bp with three SNPs (position766:
C-T; 800: T-C; 836: C-T). TaCD4 was 851 bp, with two insertions (7 and 1 bp), one
gap (4 bp) between 731bp and 778bp, and had many SNPs compared to TaCD1 and
TaCD2.
In the coding parts of the three genes, the cDNAs TaCD1 and TaCD2 had 19 SNPs
compared TaCypB-A, 17 SNPs compared to TaCypB-B, 2 SNPs with AetCypB-D and
none with TaCypB-D. TaCD4 had 10 SNPs with TaCypB-A, one SNP with TaCypB-B,
82
five SNPs with TaCypB-D and 13 more SNPs with AetCypB-D in the length of 306 bp
to 642 bp. Compared CypB from the three genomes, TaCD1 and TaCD2 has two
deletions (7 bp and 1 bp) and one insertion (4 bp) between 724 bp to 846 bp compared
to TaCypB-B; two insertions with TuCypB-A; and only five or three SNPs with
AetCypB-D. TaCD4 had only three SNPs with TaCypB-B, but exhibited
insertions/deletions in relation to TuCypB-A and AetCypB-D. TaCD1 and TaCD2 were
deduced to be from D genome and TaCD4, from B genome.
The phylogenetic tree of CypB exon contigs in wheat species and three cDNA CypB
clones from T. aestivum cv Cranbrook confirmed the genomic assignment, i.e., TaCypB-
A most likely being from A genome, TaCypB-B from B genome, TaCypB-D from D
genome; the cDNAs TaCD1 and TaCD2 being from D genome; TaCD4 from B genome
(Fig 3.7).
AetCypB-D
TaCD1
TaCD2
TaCypB-D
TaCypB-B
TdCypB-B
TaCD4
TuCYPB-A
TaCypB-A
TdCypB-A
Rice
72
100
39
81
57
30
94
32
Fig 3.7 Phylogenetic analysis of CypB exon contigs and cDNA sequences. Trees were generated by Neighbor-Joining method of the Bootstrap Test of Phylogeny in MEGA 4.0 program (http://www.megasoftware.net/mega.html), based on alignments of CypB exon contigs from seven genomic genes shown in Fig 3.3 with sequences of cDNA clones (TaCD1, TaCD2, TaCD4) in T. aestivum cv. Cranbrook and exon contig of CypB in rice (presented later) (Fig 3.8).
83
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB-B ATACGATCCGAGATGGCGATGAGGGCGTGGAGGAGGAGCGCGGCGGCGAGGG------CCCCGGCGCACCTG---------TGCCTGTGGCTGGCGCTCGTCGCCGCCACCCTGGTGCTCGCCCAGG-GC--------------AAGAAA
TaCypB-A ....................................................------..............---------..............................................-.T--------------.....G
TaCypB-D .................................C..................------..............---------..............................................-..--------------......
TdCypB-B ....................................................------..............---------..............................................-..--------------......
TdCypB-A ....................................................------..............---------..............................................-.T--------------.....G
TuCYPB-A ....................................................------..............---------..............................................-.T--------------.....G
AetCypB-D .................................C..................------..............---------..............................................-..--------------......
TaCD1 .................................C..................------..............---------..............................................-..--------------......
TaCD2 .................................C..................------..............---------..............................................-..--------------......
TaCD4 ....................................................------..............---------..............................................-..--------------......
Rice ------------......GG...C.G...........T.C......T....AGGCCGC..AT.T...G..C.GCCTCGGTC.....C...A.C.T....................C...........T..TCATCCACCTCGCG......
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB-B TCCAA------CCTGTCTGAGGTGACGCACAAGGTCTACTTCGACATCGAGATCGACGGCAAGCCCGCAGGTCGGGTTGTCATGGGACTTTTCGGCAAGGCTGTTCCTAAAACCGCAGAGAACTTCAGAGCACTCTGCACAGGTGAGAAA
TaCypB-A ..G..------......G.............................T...............................................................................................A......
TaCypB-D .....------...T................................................................................................................................A......
TdCypB-B .....------...........................................................................................................................................
TdCypB-A ..G..------......G.............................T...............................................................................................A......
TuCYPB-A ..G..------......G.............................T...................T...........................................................................A......
AetCypB-D .....------...T................................................................................................................................A......
TaCD1 .....------...T................................................................................................................................A......
TaCD2 .....------...T................................................................................................................................A......
TaCD4 .....------........................................................................................................................G..................
Rice ..G..GGCGGATT..A.......C..C..................G.......T..T..............A.......................G...A.............G............C.......T........A......
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB-B GGCATGGGCAACAGCGGCAAACCTCTCCACTACAAGGGGAGTTCGTTCCACAGAATTATCCCCAGCTTCATGATCCAAGGAGGGGACTTCACTCTTGGCGATGGAAGGGGCGGTGAATCAATCTATGGCACAAAGTTCGCCGATGAGAAC
TaCypB-A ....................G........................................................................................................C........................
TaCypB-D ..T..................................................-------------------------------------------------------------------------------------------------
TdCypB-B ......................................................................................................................................................
TdCypB-A ....................G........................................................................................................C........................
TuCYPB-A ..T.................G..............................................................T.........................................C........................
AetCypB-D ..T................................................................................C..........C...T...........T..............C........................
TaCD1 ..T................................................................................C..............T...........T..............C.............G..........
TaCD2 ..T................................................................................C..............T...........T..............C.............G..........
TaCD4 ......................................................................................................................................................
Rice ..A.CT..A..G..T......G.A.......T......A...G.A..............A........T..............T........A.....T...........T........T........G..G..............A...
84
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB-B TTCAAGCTCAAGCACACTGGACCAGGCTACCTTTCCATGGCCAATGCTGGGAGAGATACCAATGGATCCCAGTTCTTCATCACCACTGTAACCACGAGCTGGTTGGACGGCAAGCACGTCGTGTTCGGCAAGGTGTTGTCTGGAATGGAC
TaCypB-A ...........................................................................................................T..........................................
TaCypB-D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB-B ......................................................................................................................................................
TdCypB-A ...........................................................................................................T..........................................
TuCYPB-A ...........................................................................................................T..........................................
AetCypB-D ..............................................................C.............................T..............T..........................................
TaCD1 ..............................................................C.............................T..............T..........................................
TaCD2 ..............................................................C.............................T..............T..........................................
TaCD4 ......................................................................................................................................................
Rice ......A..........C.........CT...G.......................C..A..C..G........T....................C..............G.................T......C.............T
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaCypB-B GTGGTCTACAAGGTTGAGGCCGAGGGCAAGCAGAACGGGACGCCAAAGAGCAAGGTTGTCATTGCCGACAGCGGTGAAGTGCCGCTGTGATGAGACCTGAAATGGGCTCTGGTGGAA-GCTACGGTGACTAGTTTTCGTTGTAGAACCGT
TaCypB-A .......................................................................-------------------------------------------------------------------------------
TaCypB-D ------------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB-B .......................................................................-------------------------------------------------------------------------------
TdCypB-A .......................................................................-------------------------------------------------------------------------------
TuCYPB-A ..............................................................C...........A..............................T...........A...................A............
AetCypB-D ..............C..........................A...........A...........T........G..................--.......C..T...........A.................C..........G...
TaCD1 ..............C..........................A...........A...........T........G..................--.......C..T...........A.................C..........G...
TaCD2 ..............C..........................A...........A...........T........G..................--.......C..T...........A.................C..........G...
TaCD4 .....................................................................................................................-................................
Rice .....T......A....A..T......C......GT...T.A..G........A........C..G........C...C.....A...A.------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .
TaCypB-B TACTAGCCGGTCTGTTTTTTGTTTTCGTGTACTTTTGTGTGAAATTCAGACGACAAGGATTCTC----TTGCTATAAGAAAGTATAATGACTTGCGGATTTACCCGTGATCTGGCTGGCAACTTTGAAGGCATCACACACCACCGT
TaCypB-A --------------------------------------------------------------------------------------------------------------------------------------------------
TaCypB-D --------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB-B --------------------------------------------------------------------------------------------------------------------------------------------------
TdCypB-A --------------------------------------------------------------------------------------------------------------------------------------------------
TuCYPB-A ..T.GT.G..A.-------...............CC.......-..T.....G...A.C..T.TCTCT.-A......A..........A......----....T.A....A...A..............TGC..TT.TTATTTTT.
AetCypB-D ..T..T....A.-------.A.............CC.......-..T.....G....TT..T..CTCT..A......A......C..........----...............................T...............
TaCD1 ..T..T....A.-------...............CC.......-..T.....G....TT.....CTCT..A......A......C..........----...............................T...............
TaCD2 ..T..T....A.-------...............CC.......-..T.....G....TT..T..CTCT..A......A......C..........----C..............................T....T..........
TaCD4 ...................................C............................----...........................----C..............................................
Rice -------------------------------------------------------------------------------------------------------------------------------------------------- Fig 3.8 Sequence alignment of exon contigs with cDNA sequences. Exon contigs from seven genomic genes shown in Fig 3.3 with sequences of three cDNA clones (TaCD1, TaCD2, TaCD4) in T. aestivum cv. Cranbrook and exon contigs of identified CypB in rice (presented later). Alignment of TuCypB-A exon contigs up to 847bp. Dots indicate the bases identical to the first line and broken lines indicated deletion and incomplete amplification at 3’-end.
85
3.3.8 Alignment of the deduced putative CypB protein sequences in wheat species
The amino acids sequences were deduced using the ‘translate or reverse-translate’
option in Bioedit, from exon contigs of individual clones and/or genomic contigs in T.
aestivum cv. Rosella, T. turgidum ssp. durum, T. urartu, and Ae. tauschii; and three
CypB cDNAs in T. asetivum cv. Cranbrook (Fig 3.9). The sequences were aligned
using the ‘ClustalW Multiple Alignment’ in Bioedit and led to 100% identity to putative
proteins deduced from exon contigs of CypB genes in T. aestivum cv. Rosella. The
putative amino acid sequence deduced from the cDNA clone TaCD4 was also 100%
identical with that from the full-length exon contig TaCypB-B. The sequences from
cDNA clones TaCD1 and TaCD2 showed one residue variation (A131G) in relation to
TaCD4 and other genes. The proteins deduced from exons contigs of TdCypB-A and
TdCypB-B had 100% identity to each other and to comparable sections of the three
deduced proteins of common wheat cv. Rosella. The sequences deduced from contig
exons of TuCypB-A in T. urartu and AetCypB-D (clone AetC1) in Ae. tauschii had one
variant residue (TuCypB-A, position 57:A-V; AetCypB-D, position 116: L-P) which
appeared not to affect any functionally important residue.
86
60
TaCypB-B MAMRAWRRSA AARAPAHLCL WLALVAATLV LAQGKKSNLS EVTHKVYFDI EIDGKPAGRV
TaCypB-A .......... .......... .......... .......... .......... ..........
TaCypB-D .......... .......... .......... .......... .......... ..........
TdCypB-B .......... .......... .......... .......... .......... ..........
TdCypB-A .......... .......... .......... .......... .......... ..........
TuCypB-A .......... .......... .......... .......... .......... ......V...
AetCypB-D .......... .......... .......... .......... .......... ..........
TaCD4 .......... .......... .......... .......... .......... ..........
TaCD1 .......... .......... .......... .......... .......... ..........
TaCD2 .......... .......... .......... .......... .......... ..........
120
TaCypB-B VMGLFGKAVP KTAENFRALC TGEKGMGNSG KPLHYKGSSF HRIIPSFMIQ GGDFTLGDGR
TaCypB-A .......... .......... .......... .......... .......... ..........
TaCypB-D .......... .......... .......... .......... .--------- ----------
TdCypB-B .......... .......... .......... .......... .......... ..........
TdCypB-A .......... .......... .......... .......... .......... ..........
TuCypB-A .......... .......... .......... .......... .......... ..........
AetCypB-D .......... .......... .......... .......... .......... .....P....
TaCD4 .......... .......... .......... .......... .......... ..........
TaCD1 .......... .......... .......... .......... .......... ..........
TaCD2 .......... .......... .......... .......... .......... ..........
180
TaCypB-B GGESIYGTKF ADENFKLKHT GPGYLSMANA GRDTNGSQFF ITTVTTSWLD GKHVVFGKVL
TaCypB-A .......... .......... .......... .......... .......... ..........
TaCypB-D ---------- ---------- ---------- ---------- ---------- ----------
TdCypB-B .......... .......... .......... .......... .......... ..........
TdCypB-A .......... .......... .......... .......... .......... ..........
TuCypB-A .......... .......... .......... .......... .......... ..........
AetCypB-D .......... .......... .......... .......... .......... ..........
TaCD4 .......... .......... .......... .......... .......... ..........
TaCD1 .......... G......... .......... .......... .......... ..........
TaCD2 .......... G......... .......... .......... .......... ..........
213
TaCypB-B SGMDVVYKVE AEGKQNGTPK SKVVIADSGE VPL*
TaCypB-A .......... .......... .......--- ----
TaCypB-D ---------- ---------- ---------- ----
TdCypB-B .......... .......... .......--- ----
TdCypB-A .......... .......... .......--- ----
TuCypB-A .......... .......... .......... ...*
AetCypB-D .......... .......... .......... ...*
TaCD4 .......... .......... .......... ...*
TaCD1 .......... .......... .......... ...*
TaCD2 .......... .......... .......... ...* Fig 3.9 Alignment of amino acids deduced from exon contigs of the seven CypB genomic genes and CypB cDNA of three clones. Dots indicate the residues identical to the first line; broken lines indicated residues deduced from the incompletely amplified genes.
3.3.9 Comparison of putative CypB protein sequences with ER-localised Cyps in
other organisms and CypA in human and wheat
Alignment of the amino acid sequence of putative TaCypB-B deduced from exon contig
of TaCypB-B, with ER-localised Cyps in other organisms and cytosolic CypA in human
and wheat showed a very high degree of similarity except in the N- and C-terminal
regions (Fig 3.10). The sequence and secondary structure of human cyclophilin A
(hCyP-18a; Genbank NP_066953) used as reference shows three consensus sequences,
87
FHRI(V)IP(K/Q)XFMI(C)QGGDFT, (V/T)GXL(V)SMANA(R/S)GXD(N)TNGSQFFI
and WLDGKHVVFG, containing cyclosporin A-binding residues and the tetrapeptide
Ala-Ala-Pro-Ala (Hirtzlin et al., 1995). The seven residue insertion noted in Cyps of
‘organisms with chloroplasts’ (Saito et al., 1999; Johnson and Bhave, 2004b), occurring
between α-helix-II and β-strand-III, exposed as an extended loop of a domain possibly
interacting with other cellular components (Buchholz et al., 1994), was also observed in
the current analysis.
The ER-localised Cyps presented four to ten additional C-terminal residues, i.e., ELPM
in maize (AY103896) and rice, ELPL in Arabidopsis ROC7 (AtCYP20-1) and Cyp5
(AtCYP91-4) (Saito et al., 1999; Romano et al., 2004a) and SGEL in Dictyostelium
discoideum (AF123597) and Leishmania donovan (CAG33110), all of which are similar
but do not correspond to the characteristic KDEL/HDEL/HEEL ER-retention/retrieval
signal (Frigerio and Pelham, 1993; Derkx and Madrid, 2001). TaCypB-B showed a
new variant ‘EVPL’. The ten-residue motif ‘WEKPFAV(I)AKE’ in human CypB
(Price et al., 1991) and conserved in chicken, mouse and zebrafish, and the C-terminal
variable extensions in yeast and other fungal Cyps were absent in TaCypB and other
plant Cyps.
The TaCypB-B had a 40-amino-acid N-terminal extension compared to wheat CypA
(CYP18-2) (Johnson and Bhave, 2004b), which had 12% identity to the 32 amino acid
extension for ER-targeting in human CypB (Price et al., 1991). Generally this sequence
contains positively charged residue(s) (K) preceding a hydrophobic stretch, which is
thought to act as the ER-targeting signal sequence (Bose et al., 1994; Saito et al., 1999).
The ER-localised Cyp5 in Arabidopsis has a potential cleavage site of signal peptide
between A23 and K24 (Saito et al., 1999). TaCypB-B and rice CypB both exhibit a K-
rich N-terminal, with the putative cleavage site between G34-K35 or A39-K40
respectively, identical to that for Cyp5 (Saito et al., 1999).
The overall DNA identity of exon contig TaCypB-B with other ER-localised Cyps
ranged from 47% to 83%, and its amino acid identity ranged from 40% to 84%.
Sequence alignment with hCyp18-a showed TaCypB-B and rice CypB (detailed below;
section 3.3.10) had residues potentially involved in PPIase activity (Kallen and
Walkinshaw, 1992), CsA binding (Pflugl et al., 1993), calcineurin binding (Rascher et
88
al., 1998) and glycosylation (Hirtzlin et al., 1995; Pelle et al., 2002). Three residues in
TaCypB-B (R102, F107, H173) corresponded to R55, F60 and H126 in hCypA which
are highly conserved and essential to PPIase activity (Zydowsky et al., 1992) and W168
in TaCypB corresponded to W121 of hCypA essential for CsA binding (Bossard et al.,
1991). However, residue R69 shown to be essential for binding CypA-CsA complex to
calcineurin in hCyp-18a (Pelle et al., 2002) was absent in wheat and rice CypB, and
replaced by L116 in wheat and L123 in rice; this residue is also replaced in Leishmania
major Cyp by an unusual interaction residue (N) (Rascher et al., 1998). The N-linked
glycosylation sites in hCypA (N71 and N108) or in Trypanosome CypA (N81 and
N121) (Pelle et al., 2002) were only partially conserved in TaCypB-B (D118, N155).
Similarly, the O-glycosylation sites in hCypA (T73, S110) or Trypanosome CypA (T83,
S123) (Pelle et al. 2002) were not completely conserved in TaCypB-B (R120, S157).
89
TaCypB-B MAMRAWRRSA AAR-----AP AHLCLWLALV AATLVLAQGK KSN------- LSEVTHKVYF DIEIDGKPAG 58
Rice ..GSG...IP .V.RPPMSR. .SV...IV.. ....A...A. ..KAD----- .T........ .V........ 65
Maize .ETSG...R. ...ASW--S. .TVY.....A ..A.T...A. .D-------- .T........ .V........ 60
ROC7 ..S------- ---------S VT.L..SL.L LG..SAI.A. ..K-----EN .K.I...... .V.....A.. 49
AtCyp5 ..K------- ---------A SFIL.GTLFL FGAIASI.A. --------ED .K........ .V.....S.. 46
dCyp2 ---------- ---------M KVIFVV..I. LV..WAMPSE AGK------- DPKI.N..F. .....N.... 44
hCypB ---------- ---------- MKVL.AA..I .GSVFFLLLP GPSAADEKKK GPK..V.... .LR.GDEDV. 50
Mouse ---------- ---------- MKVLFAA..I VGSV.FLLLP GPSVANDKKK GPK..V.... .LQ.GDESV. 50
Chicken ---------- ---------- MKALVAATAL G-PAL.LLLP AASRADERKK GPK..A..F. .LRVGEED.. 49
Fish ---------- --MVRICERR MKFLVAVT.I VGSV.FLLFP SETEADEKKK GPK..A.... ..K.GDED.. 58
Donovan ---------- ---------- -----MRFVA VLAV..CALS F----LNVAA EP...A.... .VM..SE.L. 41
Fungi ---------- -------MTS LKSLFLSFF- LVVALGLALV N--ASEP--R GPKI.N.... ..QHGDESL. 48
Yeast ---------- ---------- MK.QFFSFIT LFACLFTTAI F--AKEDTAE DP.I...... ..NHGD.QI. 48
CYP18-2 ---------- ---------- ---------- ---------- ---------- --MANPR.F. .MTVG.A... 18
hCyP-18a ---------- ---------- ---------- ---------- ---------- --M.NPT.F. ..AV..E.L. 18
+ + + + βI βII
↓ ↓ * * * * # -*-
TaCypB-B RVVMGLFGKA VPKTAENFRA LCTG-EKGMG NSGKPLHYKG SSFHRIIPSF MIQGGDFTLG DGRGGESIYG 127
Rice .........T .......... ....-...T. K...A..F.. .A........ .......... .......... 134
Maize .I.......T .......... ....-...I. K...A..... .T........ .L........ .......... 129
ROC7 .I.......T ....V..... ....-...I. KN..A..... .......... .L......H. N.M....... 118
AtCyp5 ...I...... .......... ....-...V. K......... .K........ ........H. N.M....... 115
dCyp2 .I.F..Y..T ....V..... ....-...L. T........D .K......N. ........R. ..T....... 113
hCypB ..IF.....T ....VD..V. .A..-...F. -------..N .K...V.KD. ........R. ..T..K.... 112
Mouse ...F.....T ....VD..V. .A..-...F. -------..N .K...V.KD. ........R. ..T..K.... 112
Chicken ...I.....T ....V...V. .A..-...F. -------F.. .K...V.KD. ........R. ..T..K.... 111
Fish .I.I.....T ....T...LQ .A..-...F. -------... .K...V.KD. ........R. ..T..K.... 120
Donovan .ITI.....D A.L.T....Q ....-.H.F. -------..D .I...V.QN. ........NF ..T..K.... 103
Fungi .I.L..Y..T ..E....... .A..-...F. -------.E. .N...V.KD. ........R. ..T..K.... 110
Yeast .I....Y.LT T.Q.V...YQ .TISRDPK.. -------.LN .I...V..N. ........HR S.I..K..F. 111
CYP18-2 .I..E.YKD. ..R.V..... ....-...V. K......... .....V..D. .C......K. N.T....... 87
hCyP-18a ..SFE..ADK .......... .S..-...F. -------... .C......G. .C......RH N.T..K.... 80
βII αI αII ++ + + βIII+ + βIV +
↓ *** - - * * ↓ ** #* #
TaCypB-B TKFADENFKL KHTGPGYLSM ANAGRDTNGS QFFITTVTTS WLDGKHVVFG KVLSGMDVVY KVEAEGK-QN 196
Rice .........I ......L... .......... .......... .......... .......... .I....Q-.S 203
Maize M........I ......L... .......... .......... .......... .......... ......R-.S 198
ROC7 E......... ......F... ....Q..... .......... ....R..... ..VT...... ......N-.S 187
AtCyp5 Q......... ......V... ..S.E..... .......... ....R..... ..VQ...... .I.....-.S 184
dCyp2 K..N.....I ..SK..L... ....PN.... .......V.. ....R.T... E.IE...I.K LL.SI.S-.S 182
hCypB ER.P...... ..Y...WV.. ....K..... .......K.A .......... ...E..E..R ...STKTDSR 182
Mouse ER.P...... ..Y...WV.. ....K..... .......K.. .......... ...E.....R ...STKTDSR 182
Chicken DR.P...... ..Y...WV.. ....K..... .......K.A .......... ...E.....R ...NTKTDSR 181
Fish DR.P...... ..Y...W... ....K..... .......Q.P .......... .I.E.....R .I..TKTDGR 190
Donovan E......LNV ..-FV.A... ....PN.... ......AP.P ....R..... ...D.....L RI.KTKTNSH 172
Fungi A..K...... R..KT.L... ....K..... ......AV.P .......... E..E.Y.I.D .IQNVP.GR. 180
Yeast NT.K....DV ..DK..R... ..R.KN.... .......PCP .......... E..D.....H YI.NVKTDSR 181
CYP18-2 E.....K.VH ...K..I... ....PN.... ....C..PCN .......... E.VE.....K NI.KV.S-RS 156
hCyP-18a E..E....I. ......I... ....PN.... ....C.AK.E .......... ..KE..NI.E AM.RF.S-R. 149
βV βVI βVII αIII
TaCypB-B GTPKSKVVIA DSGEVPL--- ---------- ---------- ---- 213
Rice .S........ ....L.M--- ---------- ---------- ---- 220
Maize .Q........ ....L.M--- ---------- ---------- ---- 215
ROC7 .........V ....L..--- ---------- ---------- ---- 204
AtCyp5 .......... ....L..--- ---------- ---------- ---- 201
dCyp2 ...SKIAK.S N...L----- ---------- ---------- ---- 197
hCypB DK.LKD.I.. .C.KIEVE-- --------KP FAIAKE---- ---- 208
Mouse DK.LKD.I.V ...KIEVE-- --------KP FAIAKE---- ---- 208
Chicken DK.LKD.T.. .C.TIEVE-- --------KP FAIAKE---- ---- 207
Fish DK.LKD.S.H ...KIDVE-- --------KP FAVAKE---- ---- 216
Donovan DR.VKP.K.V A...L----- ---------- ---------- ---- 187
Fungi DR.LKD.K.V K...LEME-- --------AD VANEGDKKGS HNEL 214
Yeast NM.VKE.I.V E...LETVPL DNKDAAKLQE EIKAEASEAA HDEL 225
CYP18-2 ..CSKQ.... .C.QL----- ---------- ---------- ---- 171
hCyP-18a .KTSK.IT.. .C.QLE---- ---------- ---------- ---- 165
βVIII
90
Fig 3.10 Comparison of ER-localised cyclophilins from different species with CypA in human and wheat The aligned ER-localised cyclophilins are the putative wheat CypB protein (wCypB) deduced from exons contig of isolated CypB genomic gene (EU627095); putative rice CypB identified from rice database (http://www.tigr.org/tdb/e2k1/osa1/) (this study); ER-localized Cyps in Arabidopsis thaliana, i.e. AtCyp5 (AtCYP19-4; NM128550) and ROC7 (AtCYP20-1; NM125258); maize (AY103896); CypB (dCyp2; AF123597) in Dictyostelium discoideum; human (hCypB; M60857); mouse (M60456); chicken (M63553); zebrafish (NM213019); Leishmania donovani (CAG33110); fungi (Q5B4R3) and yeast (P35176). CypA in human (hCyP-18a; NP_066953) and wheat (CYP18-2; AY456122) used for comparisons. Dots indicate residues identical to top line; broken lines indicate gaps; arrows indicate exons (I–VII) based on the CypB genomic gene (TaCypB7B) in T. aestivum cv Rosella. Three consensus sequences containing residues for CsA binding and tetrapeptide (Ala-Ala-Pro-Ala) contact are in bold and underlined. Asterisks in bold (*) above the lines indicate residues involved in CsA contact, including the key W residue. +residues involved in enzymes activity;
#calcineurin binding residues;
−N-linked and O-linked
glycosylation sites. The seven-amino-acid insertion characteristic of ‘organisms containing chloroplasts’, the N-terminal ER-targeting sequence and the C-terminal ER-localisation signal are boxed. The putative cleavage site of signal peptide is at K35 in TaCypB-B. Secondary structure features based on hCypA. Table 3.5 Functionally important residues in the putative wheat and rice CypB proteins
Function Residues
wCypB Rice Corresponding hCypA residues; Reference
PPIase activity R102, I104, F107, Q110, A148, N149, Q158, F160,
L169, H173, A191
R109, I111, F114, Q117, A155, N156, Q165, F167,
L176, H180, A198
R55, I57, F60, Q63, A101, N102, Q111, F113, L122, H126, R144 (Kallen and Walkinshaw, 1992)
Essential for PPIase activity
R102, F107, H173 R109, F114, H180 R55, F60, H126 (Zydowsky, et al., 1992)
CsA contacting
R102, F107, M108, Q110, G119, A148, N149, A150, Q158, F160, W168, L169, H173
R109, F114, M115, Q117, G126, A155, N156, A157, Q165, F167, W175, L176, H180
R55, F60, M61, Q63, G72, A101, N102, A103, Q111, F113, W121, L122, H126 (Pflügl, et al., 1993)
Essential for CsA binding
W168 W175 W121 (Bossard, et al., 1991)
Calcineurin binding (CN-binding)
L116, K172, Q195 L123, K179, Q202 R69, K125, R148 (Rascher, et al., 1998)
Essential for CN-binding
L116 L123 R69 (Rascher, et al., 1998)
N-linked Glycosylation
D118, N155,
D125, N162, N71, N108, (Hirtzlin, et al., 1995; Pelle, et al., 2002)
O-linked Glycosylation
R120, S157 R127, S164 T73, S110 (Hirtzlin, et al., 1995; Pelle, et al., 2002)
3.3.10 Identification of putative CypB gene and its promoter sequence in rice
The BLASTn search of Rice Genome Annotation Database Assembly 2006
(http://www.tigr.org/tdb/e2k1/osa1/) using TC264488 as a query sequence (section
2.16) led to identification of the BAC of Oryza sativa ssp japonica cv. Nipponbare
containing a highly similar sequence (LOC_Os06g49480) between 29972988 and
29975061 bp on chromosome 6. Alignment of the putative CypB gene in this BAC and
its coding sequence (CDS) showed a seven-exon structure (Fig 3.6). Compared to
91
TaCypB-B, the rice gene had 54.7% identity in DNA sequence, with much higher
identity over exons (83%) than introns (30%) (Table 3.6). The putative CypB protein
encoding by its CDS contained 220 amino acids and shared 83.2% identity with wheat
putative CypB (213 amino acids) (Fig 3.10; Tables 3.6), suggesting they are
orthologous. The ‘Rice Genome Browser’ interface of TIGR Rice Genome Annotation
(http://www.tigr.org/tigr-scripts/osa1_web/gbrowse/rice/) was used to find the region
1000 bp upstream of the ATG and analysed it as putative promoter region (Appendix
III).
Table 3.6 Sequence comparisons of the isolated TaCypB7B gene and its putative protein product, with those from rice*
*LOC_Os06g49480 in TIGR Rice Genome Annotation Database (http://www.tigr.org/tdb/e2k1/osa1/, first accessed 11/06).
aLength of genes or individual exons, from start codon in exon I to stop codon
in exon VII; bnumber of amino acids encoded in the putative protein.
3.3.11 Identification of CypB promoter sequences in diploid wheat
As introduced in section 3.3.4, IPCR was applied for amplification of unknown sections
flanking TuCypB-A. Amplifications on SacI digested and circularized gDNA with first
round primers (CYPBF3-CYPBIPCR-F4), and second round PCR on these products
(with CYPBF3-CYPBIPCR-R1) gave ~2200bp products which were cloned. Clone 2
(TuD2) in T. urartu was confirmed to be of interest by amplification with primer pair
(CYPBF3/CYPBIPCR-R1) and digestion with (SacI) (Fig 3.5). The digestion showed
two bands, ~1700 bp and ~500 bp, suggesting likely inclusion of upstream and
downstream regions in this clone. The insert was sequenced and the section of 505 bp
(Genbank FJ799756) was identified as an upstream section of TuCypB-A; the other
1629 bp up to the SacI site (G’AGCTC) being as downstream.
Length (bp) Identity (%)
Rice Wheat
Exon I 114a 99
a 48.24
Exon II 79 73 39.24
Exon III 48 48 91.76 Exon IV 24 24 91.76 Exon V 183 183 85.79
Exon VI 76 76 88.16 Exon VII 139
a 139
a 84.89
Total Exons 663 642 83.25 Intron I 249 232 38.55 Intron II 396 320 22.37 Intron III 93 90 35.48 Intron IV 466 827 17.44
Intron V 108 107 30.68 Intron VI 99 96 37.37 Total Introns 1411 1672 30.31 Total 2074
a 2393
a 54.72
CDS 663a 642
a 83.25
Protein 220b
213b
83.18
92
The putative promoter of AetCypB-D in Ae. tauschii was amplified by IPCR from SacI
digested and circularised gDNA with the primer pairs noted above. The size of PCR
products with CYPBF3-CYPBIPCR-R1 was ~3000bp (Fig 3.11A). Clone 4 was
confirmed (by PCR with CYPBF3-CYPBIPCR-R1) to contain the expected size insert.
Digestion of this PCR product with SalI showed two bands (~2800 bp, ~200 bp) (Fig
3.11B). The insert was sequenced partially and the 1216 bp section (FJ799757) was
identified as upstream of AetCypB-D. The putative promoters TuCypB-A (P) in T.
urartu and AetCypB-D (P) in Ae. tauschii were aligned with that of rice CypB
(Appendix IIB). Alignments of 500 bp areas showed 75.9% identity over 500bp region
and 88.5% identity over the 370 bp region to the start codon.
A B
Fig 3.11 Section CYPBIPCR-R1/CYPBF3 amplified by inverse PCR from SacI digested and circularised genomic DNA (A) in Ae. tauschii and confirmation of the clones with expected insertion (B) Arrow in A indicates the isolated section of CYPBIPCR-R1/CYPBF3. Arrows in B indicate the right sizes of PCR products and digestion by SacI. Lane 1 in B: PCR products by primer pairs CYPBIPCR-R1/CYPBF3; Lane 2 in B: digestion of PCR products by SacI.
The main regulatory elements identified are listed in Table 3.5 and Appendix III. The
potential TATA-box motif (TAAATAAA) for determination of transcription start site
(TSS) was identified in rice CypB promoter at -484bp from the start codon and two
CAAT box motifs noted downstream of it, CGCAAT (-276) and CTCAAT (-245). No
TATA and CAAT boxes were found for TuCypB-A and AetCypB-D, whereas GC-rich
motifs critical to transcription initiation (Pugh and Tjian, 1991; Sargsyana et al., 2002)
occurred at –114 in rice CypB, -180 in TuCypB-A, and -183 in AetCypB-D. In addition,
93
the E-boxes (CACCTG) considered responsible for tissue specific expression of napA in
transgenic Brassica seeds (Stalberg et al., 1996) and puroindoline (Pin) genes in wheat
seeds (Simeone et al., 2006) were present in all three. Motifs similar to the AT-rich
motif (ATATTTAT/AA/T) and the soybean embryo factor (SEF) binding sites for seed-
specific enhancer (Allen et al., 1989) present in seed storage protein gene promoter of
Coffea arabica (Marraccini et al., 1999) were identified at -343 and -489 of rice CypB,
-314 in TuCypB-A and -318 of AetCypB-D. The Dof core sequence (AAAG) for light
regulation and tissue specific expression of maize storage protein zein (Vicente-
Carbajosa et al., 1997) and wheat Pin genes (Lillemo et al., 2002; Simeone et al., 2006)
appeared frequently, and motifs similar to the endosperm consensus motif (prolamin
box) (TG(T/A/C)AAA(A/G)(G/T)) considered responsible for endosperm specific
expression of wheat storage proteins (Colot et al., 1987) and Pins (Lillemo et al., 2002;
Simeone et al., 2006) also occurred in all three. The dyad repeats (CAAN2-9TTG)
related to endosperm specific expression of barley chitinase (Leah et al., 1994) and
wheat Pin (Lillemo et al., 2002) were at -468 of rice CypB and similar sequences
occurred at -495 (CAAN12TGG) and -201 (CAAN11TGG) of TuCypB-A and AetCypB-
D. The CT-leader box (CTCTCTCT) influencing gene expression (Marraccini et al.,
1999) or similar motifs also occurred in all three. Sequences up to -370bp upstream of
start codons thus appear sufficient for transcription initiation of these genes. Further,
the ER stress responsive element (ERSE-II) motif (ATTGG-N-CC(A/C)N(C/G/T)
(Yoshida et al., 1998; Kokame et al., 2001) was identified at position -954.
94
Table 3.7 Putative regulatory elements in the promoters of CypB genes in wheat and rice*
Regulator element
Consensus sequence Position; gene
Proposed function
Reference
CT leader box
CTCTCTCT CTCTCTCCCTCTCTTTCTCTCT CTCTCTCTCTCT CTCTCTCCCTCTCTTTCTCTCT
-49, R -34, A -44, D
Influence gene expression
quantitatively
Marraccini et al., 1999
Endosperm motif (Prolamin-box)
TG(T/A/C)AAA(A/G)(G/T) TGAAAAGA TATAAAGT TGCAAAGC TATAAAGT TGCAAAGC
-368, R -273, A -205, A -276, D -208, D
Endosperm-specific
expression
Colot et al., 1987; Lillemo et al., 2002; Simeone et al., 2006
AT-rich motif ATATTTAT/AA/T AAAATTTTTAT TTTTTTAAATAAA ATTTTTTAATCATTTTTT ATTTTTTTAATCATATTTT
-343, R -489, R -314, A -318, D
Seed-specific enhancer
(soybean embryo factor (SEF) binding site)
Allen et al.,1989; Marraccini et al., 1999
GC-rich motif
TCCCGCCCCTAACTCCGCCCA TGGGCAGAGTGCGGAGACCGC TGCGGTCGGGGGCGAGCAGCC TGCAGTCGGGGGCGAGCAGCT
-114, R -180, A -183, D
Critical for TSS Pugh et al., 1991 Sargsyana et al., 2002
Dof Core AAAG
frequent Light regulation, tissue specific
gene expression
Vicente-Carbajosa et al., 1997; Lillemo et al., 2002; Simeone et al., 2006
E-box CANNTG CACCTG CACCTG CACCTG
-169, R -125, A -133, D
Tissue specificity, developmental control
Stålberg et al., 1996; Simeone et al., 2006
Dyad repeats
CAA(N)2-9TTG CAAAAAGTATTTG CAAATTTTTG CAAAATACTTG
-468, R -495, A -464, D
Endosperm specificity
Leah et al., 1994; Lillemo et al. , 2002
*Consensus sequences (shown in parentheses in bold) of reported regulatory elements, and their positions upstream (-) of the start codons of the putative CypB genes of rice (R) and two diploid wheat progenitors, TuCypB-A of T. urartu (A) and AetCypB-D of Ae. tauschii (D).
3.4 Discussion
3.4.1 Wheat cyclophilin B genes show significant diversity in introns
Three different CypB genomic copies have been isolated and sequenced from T.
aestivum cv Rosella. Alignment of the full-length sequence TaCypB-B with the isolated
cDNA clones indicated the genes contain seven exons and six introns, with total length
of ~2.6kb. To our knowledge, this is the first complete sequence of a cereal CypB gene.
The results show a complete conservation of exons in size with single nucleotide
polymorphisms (SNPs) mainly in exon II and V (Figs 3.3, 3.6). Major variations
existed in introns including insertions or deletions in introns I, II, IV, V and VI, and/or
SNPs in all introns. The variations noted in intron sequences provided the basis for
assignment of the genes in different genome and physical locations on chromosomes.
95
3.4.2 Genome assignment of cyclophilin B genes supports the origins of A genome
and D genome from T. urartu and Ae. tauschii
The CypB genes were also isolated and sequenced from the tetraploid (T. turgidum ssp.
durum) and diploid (T. urartu and Ae. tauschii) progenitors of wheat. Two copies were
identified from durum and one each from T. urartu and Ae. tauschii. Alignment of
genomic sequences (TaCypB-A, TaCypB-B, TaCypB-D) of T. aestivum cv Rosella with
those from T. urartu (TuCypB-A) and Ae tauschii (AetCypB-D) showed identities of
<90% between the three common wheat genes; however, their identities with TuCypB-A
and AetCypB-D were 99.7% and 99.8%, respectively. Further, the phyllogenetic tree of
CypB exon contigs and cDNAs confirmed that TaCypB-A was most related to the A
genome, TaCypB-B to the B and TaCypB-D to the D genome. The data supports the
contention that the A genome of 6n and 4n wheats may have been contributed by T.
urartu (Sears, 1969; Breiman and Graur, 1995) and D genome of 6n by Ae. tauschii
(Gill and Kimber, 1974).
3.4.3 Section(s) of Cyclophilin B genes may be divergent in D genome of common
wheat compared to its progenitor
From the lack of success in amplifying the full-length TaCypB-D gene of common
wheat with the primer CYPBR9, it may be deduced that this gene is partial in common
wheat or that the area of primer binding may be divergent. Further, from two hints (i)
primer sequence corresponding to CypBR9 exists in AetCypB-D; (ii) its full length
(WC1-CYPBR9) cDNA clones (TaCD1, TaCD2) could be obtained in cv. Cranbrook, it
is hypothesised that the 5’ area of the gene may be deleted/divergent in Rosella. The
hypothesis can be tested by amplification of the TaCypB-D gene's 3’ and 5’ flanking
sequences by IPCR.
3.4.4 Putative wheat CypB protein shows potential roles for ER protein regulation
The putative wheat CypB protein deduced from the exon contig of TaCypB-B is
comprised of 213 amino acids, with an estimated molecular mass of ~23kDa. The 40
residue N-terminal sequences are not conserved, with identities of 9% (wheat to
Arabidopsis) and 12% (wheat to human); however, the reported cleavages sites (Saito et
al., 1999) appear conserved in all ER-localised Cyps in plants, e.g. G-K in wheat and A-
K in rice, maize and Arabidopsis (Fig 3.10). The wheat CypB contains the putative N-
terminal signal for ER-targeting (Price et al., 1991) and the mature protein in ER would
96
be 19.2 kDa. The C-terminal tetrapeptide signal EVPL is unusual, but has acidic and
hydrophobic residues and partially corresponds to ELPM from maize, ELPL from
Arabidopsis, SGEL from Dictyostelium discoideum and Leishmania Donovan, and the
reported C-terminal ER-localization signals KDEL, HDEL or HEEL (Price et al., 1994;
Frigerio et al., 1993; Derkx et al., 2001), but not to the 10-residue signals
VEKPFAIAKE from animals including human CypB (Price, et al. 1991). The 19 kDa
protein (AtCYP19-4) (with the N-terminal signal) in Arabidopsis has a role in
regulating the protein GNOM required for coordination of cell polarity along the
embryo axis (Grebe et al., 2000). Although the amino acid sequence of wheat TaCypB-
B exhibits an overall identity of only 50.2% with human hCypB, the wheat protein
exhibits all functionally residues including the potential CsA and calcineurin contact
residues, the Trp essential for CsA-binding, and the PPIase catalytic residues as reported
from human cytosolic CypA and ER-localised CypB (Pflügl et al. 1993; Price et al.,
1991). The four residues Arg55, Phe60, His126, W121 in human CypA (hCyp18-a)
essential for PPIase activity and CsA contact (Zydowsky et al., 1992) are conserved in
all the aligned ER localised Cyps in plants, e.g. ER-localised Cyps (AtCYP19-4 and
AtCYP20-1) in Arabidopsis, purified maize microsomal Cyp, putative rice CypB
identified and other organisms (Fig 3.10). The N-glycosylation site in TaCypB-B is
partially conserved and O- glycosylation site isn’t conserved, compared to reported ones
in Trypanosome CypA (Pelle et al., 2002), so residues D118, R120 and S157 in
TaCypB-B may be essential for glycosylation.
The seven-residue insertion of unknown function noted here is a characteristic of Cyps
from ‘organisms containing chloroplasts’ (Saito et al., 1999; Johnson and Bhave,
2004b), occurring between α-helix-II and β-strand-III, exposed as an extended loop of a
domain possibly interacting with other cellular components (Buchholz et al., 1994). It
isalso proposed to be located in a domain interacting with other proteins and thus might
be relevant to functions reported for Cyps involving protein-protein interactions (Galat,
1993). The 19 kDa AtCYP19-4 in Arabidopsis has a role in regulating the protein
GNOM required for coordination of cell polarity along embryo axis (Grebe et al., 2000).
The ER-localised CyP in human, with prolactin complex, acts as a transcriptional
inducer (Rycyzyn and Clevenger, 2002) and also cooperates with other chaperones
(PDI, Bip, Grp94) in playing crucial roles in protecting cells against ER stress (Kim et
al., 2008). Wheat CypB shows all functional residues as well as N-terminal ER-
97
targeting and C-terminal ER-retention sequences. TaCypB-B shares 83.8% identity
with CypB deduced from maize cDNA (AY103896) and a 30-amino-acid sequence
(K38-F67; Fig 3.10) of microsomal cyclophilin B protein of maize (Sheldon and Venis,
1996). The present observations thus indicate that the wheat CypB is possible to be ER-
localised and play important enzymatic roles in storage protein processes and in
addition may be a chaperone for other partners and roles.
3.4.5 Wheat CypB has orthologues in rice and other species
The wheat CypB orthologue in rice was identified due to their high identity and both
show similar gene structures (Fig 3.6). The wheat gene shows low identity (<50%) in
exons I and II compared to rice, but much higher (~85%) from exon III to VII,
indicating the functionally important sections (e.g., catalytic and CsA contact sites) are
encoded in the latter areas. TaCypB-B shares 69% and 67% identity on coding
sequence level with genes encoding the ER-localised AtCYP19-4 and AtCYP20-1,
suggesting it has at least two orthologues in Arabidopsis. Alternatively, five ER-
localised Cyps (AtCYP19-4, AtCYP20-1, AtCYP21-1, AtCYP21-2, AtCYP23) occur in
Arabidopsis (He et al., 2004; Romano et al., 2004a) and two in human (Galat, 2004);
thus more than one type of Cyp may be involved in protein processes occurring in the
ER of cereals.
3.4.6 The promoter elements suggest CypB proteins may be under tissue and stress
specific regulation in cereals
The promoter sequences of the putative rice and wheat CypB genes contain conserved
regulatory sequences such as GC-rich motif critical for initiation of transcription (Pugh,
et al., 1991; Sargsyan et al., 2002), the E-box (Stålberg et al., 1996; Simeone et al.,
2006) and Dof core (Vicente-Carbajosa et al., 1997; Lillemo et al., 2002; Simeone at al.,
2006) for tissues specific expression, endosperm motif (prolamin-box) (Colot et al.,
1987; Lillemo et al., 2002; Simeone et al., 2006) and dyad repeats for endosperm
expression (Leah et al., 1994; Lillemo et al., 2002), and AT-rich motif for seed-specific
enhancer (Allen et al.,1989; Marraccini et al., 1999). These results support the tissue-
specific up-regulation of Cyp genes noted in developing wheat endosperm (Grimwade
et al., 1996). High levels of storage protein expression are suggested to be an internal
stress (Hara-Nishimura, et al. 2004), and presence of ER stress responsive element
(ERSE-II) in the rice promoter (Yoshida et al., 1988; Kokame et al., 2001) also supports
98
the suggested role of ER-Cyps in stress response (Derkx, 2001; Joseph, 1999). Further,
the tissue specific elements noted strongly suggest the CypB in wheat and rice may play
important role in storage protein folding/sorting processes and against ER-stress.
In order to address the roles of CypB, further work can be pursued such as; (i) gene
expression studies under stress conditions and during seed development; (ii) testing of
CypB location in ER experimentally by immunology techniques; (iii) testing of CypB
interactions with storage or other proteins by yeast-two hybrid or similar systems or
other biochemical studies; (iv) study of direct roles in deletion lines in Arabidopsis or
other plants; (v) experimental testing of promoter sequences; (vi) the numbers of ER- or
other Cyps can be addressed through genome, transcriptome or proteome databases.
The genomic and cDNA clones and all sequence data obtained here are most essential
for studies of gene regulation, physical and genetic mapping, as well as assessing any
association with grain quality and other QTLs, and as a potential marker for such traits.
Many of these aspects are addressed in the next two chapters.
99
Chapter 4
PHYSICAL AND GENETIC MAPPING OF
CYCLOPHILIN B GENES
100
4 Physical and genetic mapping of cyclophilin B genes
4.1 Abstract
Molecular characterisation of CypB genes and their predicting promoter sequences in
wheat and rice are the important prerequisites for understanding the functions of their
encoding proteins and essential tools for physical mapping and genetic mapping of
thesis genes. The aims of the work described in this chapter were determination of
physical and genetic maps of the CypB genes and identification of any co-localised
and/or associated QTLs related to grain quality and other traits. Using variable
restriction sites to develop RFLP assays, the genes were localised on chromosomes
7AL, 7BL, and 7DL, in agreement with the rice genome project ‘wheat bin mapped
markers’ data. AFLPs were identified for TaCypB7A between the mapping parental
lines Tasman x Sunco. RFLP digested by the enzyme (HpyCH4IV) was identified for
TaCypB7B between the mapping parental lines Tasman x Sunco, CD87 x Katepwa, Janz
x Kukri. No polymorphism was identified for partial TaCypB7D gene on sequence
level between the ten mapping parental lines. The marker for TaCypB7A was used to
screen double haploid progeny lines of Tasman x Sunco cross revealing no co-
segregation with other markers due to the limited markers on chromosome 7AL. On the
other hand, the marker for TaCypB7B was found to be linked with several other markers
and gene Stb8 on chromosome 7B by screening the same progeny.
101
4.2 Introduction
Alignment of the putative CypB amino acid sequences in wheat with other ER-localised
Cyps showed it contained all the features of ER Cyps (chapter 3). ER-localised Cyps
are most directly relevant to the processes of storage protein folding and formation of
protein bodies by both routes (see section 1.2.4). Characterisation of the CypB genes in
T. aestivum and its progenitors as well as rice and polymorphisms of genes from
different genomes provided the essential data for: (1) allele specific primer design (2)
prediction of chromosomal locations of CypB genes in wheat, (3) analysis of these
genes in ten parental lines (Tasman x Sunco, CD87 x Katepwa, Janz x Kukri,
Cranbrook x Halbred, Egret x Sunstar) as well as their doubled haploid lines generated
as part of the Australian National Wheat Molecular Marker Program (NWMMP)
(Kammholz et al., 2001).
The aims of this chapter were to (1) identify wheat orthologues at the rice CypB locus
and their chromosomal position; (2) generate molecular markers for CypB genes for
physical mapping and genetic mapping; (3) search any QTLs on the CypB locus in rice
and wheat associated with CypB genes.
4.3 Results
4.3.1 Identification of putative wheat orthologues of the rice cyclophilin B gene and
their chromosomal positions
The putative rice cyclophilin B (LOC_Os06g49480) reported in chapter 3 was used to
identify any potential wheat bin markers at this locus. The ‘Rice Genome Browser’
interface of the TIGR Rice Genome Annotation (http://www.tigr.org/tigr-
scripts/osa1_web/gbrowse/rice/) was used to identify the region that included the rice
‘LOC_Os06g49480’ and select ‘Wheat Bin Mapped Markers’ on the track feature.
Three markers were identified, i.e., KSU027BE590822, NDS021BE406148, and
UNL036BF200636 (Fig 4.1). In order to identify the cDNA probes used for physical
mapping experiments, which are available in the GrainGenes structured query language
(SQL) database. The identified markers were entered into the ‘Mapped Loci for EST-
derived Probes’ query in this database (http://wheat.pw.usda.gov/cgi-
bin/westsql/map_locus.cgi). This showed that KSU027BE590822 had been mapped to
chromosome 1 (1BL: 2-0.69-0.85, 1DL: 2-0.41-1.00, and 1AL: 3-0.61-1.00) by
102
Southern blotting, by two probes (5’ probe: BE590822, 3’ probe: CD453728);
UNL036BF200636 to chromosome 1 (1AL: 3-0.61-1.00, 1BL: 3-0.85-1.00) by 5’
probe BF200636 and 3’ probe CD453641, and NDS021BE406148 to chromosome 7
(7AL: 1-0.39-0.71, 7DL: 2-0.61-0.82, and 7BL7-0.63-0.78) by a 5’ probe BE406148
(Appendix IV). Sequences of the 5’ probes (BE590822, BF200636, BE406148) were
obtained from the EST database (http://www.ncbi.nlm.nih.gov/Database/), aligned with
exon contig of TaCypB-B and showed 97.2%, 98.1%, and 99.5% identities, respectively,
in the overlapping sections.
Fig 4.1: Putative wheat orthologues at the CypB locus of rice. The 2.478kb region of rice chromosome 6 contains the putative CypB gene (BAC LOC-Os06g49480: 29,972,847 to 29,975,324). Three wheat bin mapped markers were found: KSU027BE590822-3 on wheat Chr 1, NDS021BE406148-1 on wheat Chr 7*, and UNL036BF200636-4* on wheat Chr 1 (http://www.tigr.org/tigr-scripts/osa1_web/gbrowse/rice).
4.3.2 Development of molecular markers for physical mapping
PCR-based amplified fragment length polymorphism (AFLP) followed by restriction
fragment length polymorphism (RFLP) assays for specific enzymes were designed
based on the variations in introns of the three CypB genes of T. aestivum cv. Rosella
(Figs 3.3, 3.6). The expected sizes of WC3-CYPBR5 gene sections in T. aestivum, cv.
Rosella were 1049 bp from the A, 1073 bp from B and 1045 bp from D genome. Using
the “Restriction Map Program” in Bioedit, maps were predicted for these sections for 4
and 6 base cutter enzymes (Fig 4.2). Four enzymes (HaeIII, MscI, Stul, Spel) were
identified to be specific to TaCypB-A. Five (Psil, BsIGI, ACII, HpyCH4IV, NcoI) were
specific to TaCypB-B and none specific to TaCypB-D. HaeIII and NcoI were selected
for double digestion to generate RFLPs (detailed later). Based on genomic sequences,
HaeIII was expected to cut the 1049bp section of TaCypB-A twice, at 261 bp and 857
bp, to produce 596+261+192 bp bands, NcoI would cut the 1073 bp section of TaCypB-
103
B to generate 690+383 bp bands, but neither would cut the 1045 bp section of TaCypB-
D.
AclI
BsrGI
BstZ17I
HinP1I HhaI
HpyCH4IVTaiIPsiI
NcoI
PsiI
ScaI
BspHI
HaeIII
MscI
HaeIII
StuI
ScaISpeI
TaiIXhoI
TaqI
BspHI
HinP1I HhaI
TaqI TaqI
XhoI
TaqI
Exon IIIIntron IIIExon IV
Intron IV
TaCypB-A TaCypB-B TaCypB-D
Exon V
Fig 4.2 Deduced restriction maps of Exon III to Exon V sections of TaCYPB-A, TaCYPB-B and TaCYPB-D genes from common wheat. The restriction sites were predicted using the Bioedit program. Enzymes sites unique to a gene are shown in red.
4.3.3 Experimental physical mapping of wheat Cyp B genes
In order to experimentally determine the location of isolated genomic CypB genes on
wheat chromosomes, PCR amplifications were conducted in two rounds. First
amplifications were conducted with the primer pair WC1-CYPBR5 (Table 4.1; Fig 4.3)
on gDNA of the 21 nullisomic-tetrasomic lines. Nested PCRs were then conducted on
first round products with WC3-CYPBR5 (Table 4.1; Fig 4.3) (data not shown).
104
Table 4.1 Primer pairs used for physical and genetic mapping*
Forward primer Reverse primer AT Expected Length
c
Purpose
WC1 (114-131bp)a
ATACGATCCAAGATGGCG CYPBR5 (411-430bp)a CTGTGGAACGAACTCCCCTT
57oC
~1800bp
First round PCR
WC3 (299-316bp)a
TCGGGTTGTCATGGGACT3’ CYPBR5 (411-430bp)
a
CTGTGGAACGAACTCCCCTT 55oC
~1000bp
Physical Mapping
CYPB-A1(625-643bp)b
CCTGCACTGCACCTAATCA CYPBR-A1 (1737-1754bp)
b
CTT GCC GCT GTT GCC CAT 57oC
1130bp
A genome
CYPBF4 (84-98bp)b
CGTCGCCGCCACCCT CYPBR-B1(1770-1789bp)
b
GGCATGGTCAGCAACCAGTG 62oC
1706bp
B genome
CYPB-D1 (502-519bp)b
CCAATATCGCATGCCTGT CYPBR-D1(1730-1750bp)
b
GATTAAGTGGGGCATGGTCAG 54oC
1249bp
D genome
*Table 2.7 partly reproduced for convenience. a
Primer positions based on TC264488. b
Primer positions based on genomic sequence data obtained during this project. Bold red letters in allele specific primers indicate specificities to genomes.
cExpected length based on genomic
sequence data introduced in chapter 3. AT: Annealing temperature.
242 369 90 851 106 93 TaCypB-B48 24
232 320 90 827 107 96 TaCypB-A
224 366 90 823 TaCypB-D
111 73 183 76 136
111 73 183 76 120
111 73 61
20448 24
48 24
48 24
WC1
WC1
WC1
CYPBR5
CYPBR5
CYPBR5
CYPBF4 CYPBR-B1
CYPB-A1 CYPBR-A1
CYPB-D1 CYPBR-D1
WC3
WC3
WC3
Fig 4.3 Positions of primers for physical and genetic mapping of the genomic CypB genes Forward and reverse arrows indicate forward and reverse primers, respectively. Arrows pointing down indicate HaeIII sites; arrow pointing up indicated Ncol site (see section 3.3.5).
All WC3-CYPBR5 PCR products were double-digested with HaeIII+NcoI. The results
showed the lines N7A-T7D, N7B-T7D and N7D-T7B lacked the bands expected for A,
B or D genomes, respectively, while all other lines including control (CS: Chinese
Spring) showed six bands from all genes (results for D genome chromosomes 1 to 6
shown in Fig 4.4A; data for other lines not shown), indicating the genes are located on
chromosome 7A, 7B, and 7D. The same test was then applied to ten deletion lines of
chromosome 7 from CS (Endo and Gill, 1996) (Fig 4.4B). The lines 7AL-1, 7BL-2,
7BL-7, 7DL-2, 7DL-3 and 7DL-5 lacked the respective bands. Based on deletion
breakpoints (FL: fraction length, indicates location of deletion breakpoint), the genes
appeared to be located on (i) 7AL-1 towards the distal region of the long arm (FL: 0.39-
1.00); (ii) 7BL-7 towards the distal region of the long arm (FL: 0.63-1.00); (iii) 7DL-3
towards the distal region of the long arm (FL: 0.82-1.00) (http://www.k-
state.edu/wgrc/Germplasm/Deletions/grp7L.html). Following this work, the genes were
designated as TaCypB7A, TaCypB7B and TaCypB7D (Fig 4.5).
105
Fig 4.4 Physical mapping of CypB genes using RFLPs (A) Nullisomic-tetrasomic lines in Chinese spring background (CS) and control DNA (CS). (B) Deletion lines of chromosome 7 and control (CS). PCR products were amplified from gDNAs with WC3/CYPBR5, digested with HaeIII+NcoI and electrophoresed on 1.7% agarose gels. Fig 4.5 Physical mapping of three CypB genes from wheat Red arrows: CypB genomic gene locations, i.e. TaCypB7A located in 7AL-1 towards the distal region of the long arm of 7A, TaCypB7B located in 7BL-7 towards the distal region of the long arm of 7B, and TaCypB7D located in 7DL-3 towards the distal region of the long arm of 7D chromosome. Deletion breakpoints as reported in Endo and Gill (1996) (http://www.k-state.edu/wgrc/Germplasm/Deletions/grp7L.html, last accessed 04/2009). The numbers on the left indicate C-banded and unbanded regions and outlined numbers indicate C-bands that did not differentiate well.
TaCypB7A
TaCypB7B
TaCypB7D
106
4.3.4 Confirmation of PCR products amplified with allele specific primers from
different genomes in T. aestivum cv. Rosella
The amplification of partial CypB gene from different genomes of gDNA of T. aestivum
cv. Rosella was conducted by nested PCR. First round PCR was with WC1/CYPBR5
(data not shown), followed by second round on these products with allele specific
primer pairs CYPB-A1/CYPBR-A1 (for A genome), CYPBF4/CYPBR-B1 (B genome)
and CYPB-D1/CYPBR-D1 (D genome) (Table 4.1) with expected sizes of ~1100bp,
~1700bp and ~1250bp, respectively (Fig 4.6). Based on sequence data of the genes in
cv. Rosella (Chapter 3), HaeIII presents different digestion patterns in the sections
amplified by allele specific primers, i.e., two sites in CYPB-A1/CYPBR-A1 (expected
sizes 160bp, 374bp, 596bp after digestion); one in CYPBF4/CYPBR-B1 (expected sizes
363bp, 1343bp after digestion) and none in CYPB-D1/CYPBR-D1. In order to confirm
that the PCR products were from different genomes. The products generated by allele
specific primers were thus also digested by HaeIII and the results coincided with
expecting patterns (Fig 4.6).
Fig 4.6 PCR products amplified with allele specific primers from different genome of gDNA in T. aestivum, cv. Rosella and their RFLPs digested by enzyme (HaeIII). A: PCR products amplified from A genome by primer pair (CYPB-A1/CYPBR-A1), B genome by primer pair (CYPBF4/CYPBR-B1), and D genome by primer pair (CYPB-D1/CYPBR-D1); B: PCR products digested by HaeIII and electrophoresed on 1% gels.
107
4.3.5 Identification of polymorphisms in TaCypB7A gene from ten parental lines
The CypB genes from the five pairs of parental lines (Cranbrook x Halberd, Tasman x
Sunco, CD87 x Katepwa, Janz x Kukri, Egret x Sunstar) used in the Australian National
Wheat Molecular Marker Program (NWMMP) (Kammholz et al., 2001) were amplified
by nested PCR. First round PCR was conducted on gDNAs of each parent with WC1/
CYPBR5 (data not shown). Second round PCR was on these products with allele
specific primer pair CYPB-A1/CYPBR-A1 (Fig 4.7A). The PCR products from
Tasman were larger (~1350 bp) than those of other 9 lines with the expected size
(~1100 bp). The PCR products from Tasman and Sunco were sequenced directly and
showed an insertion of 192 bp in intron IV in Tasman (Fig 4.8, Appendix IIC). A
marker for TaCypB7A could thus be developed for Tasman x Sunco progeny for genetic
mapping.
BA C
1500bp 1030bp
2000bp 1500bp
1500bp 1200bp
Cr Ha Ta Sc Cd Ka Ja Ku Eg Ss Cr Ha Ta Sc Cd Ka Ja Ku Eg Ss Cr Ha Ta Sc Cd Ka Ja Ku Eg Ss
Fig 4.7 Partial CypB PCR products with allele specific primers from the A genomes (A), B genome (B), D genome (C) in ten parental lines. Cr: Cranbrook, Ha: Halberd, Ta: Tasman, Sc: Sunco, Cd: CD87, Ka: Katepwa, Ja: Janz, Ku: Kukri, Eg: Egret, Ss: Sunstar. PCR products were electrophoresed on 1% gels.
Fig 4.8 Structure of partial CypB genes (partial Intron II to partial Exon V) in Tasman and Sunco.
108
4.3.6 Identification of polymorphisms in TaCypB7B gene from ten parental lines
The gene on chromosome 7B in the ten parental lines was amplified by nested PCR,
with the first round PCR with WC1/CYPBR5 (as per sections 4.3.4, 4.3.5) (data not
shown), followed by the allele specific pair CYPBF4/CYPBR-B1 (Fig 4.7B). All lines
showed an identical size of ~1700bp, corresponding to the expected 1706 bp. In order
to identify any variations, the PCR products were sequenced directly. Alignment of
their partial sequence with corresponding section of TaCypB7B showed an SNP (T-C) at
its position 877bp in five of the parental lines (Fig 4.9), altering the digestion site of
HpyCH4IV (A’CGT). The SNP (T-C) altered the digestion site of HpyCH4IV (A’CGT).
Enzyme (HpyCH4IV) was applied to digest the PCR products generated with
CYPBF4/CYPBR-B1 (Fig 4.10). The result showed two types, i.e. the parental lines
Tasman, CD87, Janz, Egret and Sunstar contained three bands (~650bp, ~550bp,
~500bp), while Cranbrook, Halbred, Sunco, Katepwa and Kukri contained five bands
(~640bp, ~480bp, ~370bp, ~260bp, ~80bp). A molecular marker for TaCypB7B could
thus be applied, involving PCR followed by digestion by HpyCH4IV, for progeny lines
from Tasman x Sunco, CD87 x Katepwa, and Janz x Kukri for genetic mapping.
A
870 880 890
...| ....|....| ....|....|
TaCypB7B TTAA AAACCATGTA CTCATCTATT
Cranbrook TTAA AAACCACGTA CTCATCTATT
Halbred TTAA AAACCACGTA CTCATCTATT
Tasman TTAA AAACCATGTA CTCATCTATT
Sunco TTAA AAACCACGTA CTCATCTATT
CD87 TTAA AAACCATGTA CTCATCTATT
Katepwa TTAA AAACCACGTA CTCATCTATT
Janz TTAA AAACCATGTA CTCATCTATT
Kukri TTAA AAACCACGTA CTCATCTATT
Egret TTAA AAACCATGTA CTCATCTATT
Sunstar TTAA AAACCATGTA CTCATCTATT
B
Fig 4.9 Representative SNP in CypB genes from parental pair CD87 and Katepwa)(A), alignment of sequences (867- 890bp) in parental lines with TaCypB7B (B). The base representing SNP, i.e. ‘T’ in CD87, ‘C’ in Katepwa is framed.
109
Fig 4.10 RFLPs of partial CypB gene from B genome in ten parental lines. Products of first round PCR of gDNA with primer pair WC1/CYPBR5 were amplified with. CYPBF4/CYPBBR-B1. These were then digested with HpyCH4IV and electrophoresed on 1.7% agarose gels. Cr: Cranbrook, Ha: Halberd, Ta: Tasman, Sc: Sunco, Cd: CD87, Ka: Katepwa, Ja: Janz, Ku: Kukri, Eg: Egret, Ss: Sunstar.
4.3.7 Identification of polymorphisms in TaCypB7D gene from ten parental lines
The CypB genes on chromosome 7D in the ten parental lines were amplified by nested
PCR, first round with WC1/CYPBR5 (data not shown) and second round with the allele
specific pair CYPBD1/CYPBR-D1 (Fig 4.7C). All products were same size (~1300bp),
coinciding with the expected size of 1249 bp. They were sequenced directly but lacked
variations (data not shown). Thus a marker could not be developed for the gene on
chromosome 7D.
4.3.8 Genotyping of cyclophilin B markers in mapping populations
The TaCypB7A marker was amplified from gDNA from the 162 double haploid progeny
lines (of Tasman x Sunco as described above (first round with WC1/CYPBR5, second
round with CYPB-A1/CYPBR-A1) (Fig 4.11). Eighty-four lines exhibited the AFLP
phenotype of Tasman (1350 bp), 77 lines exhibited that of Sunco (1100 bp), and one
line (#51) showed no product. DNA of four lines (#60, #71, #73, #108) was
unavailable.
110
M 1S 2T 3S 4S 5S 6S 7T 8T 9T 10S 11T 12S 13S 14S 15T 16T 17T 18T
1500bp 1030bp
M 19S 20S 21S 22T 23S 24S 25T 26T 27S 28T 29S 30T 31T 32T 33T 34T 35T 36S
1500bp
1030bp
1500bp
1030bp
M 37T 38S 39S 40S 41T 42T 43S 44S 45S 46S 47S 48S 49S 50T 52T 53S 54S
M 55S 56T 57T 58S 59S 61T 62T 63T 64S 65S 66T 67S 68S 69S 70T 72S 74T
1500bp
1030bp
111
M 75S 76S 77S 78S 79S 80T 81S 82T 83S 84T 85T 86S 87T 88S 89S 90T 91T 92S
1500bp
1031bp
1500bp
1030bp
M 93S 94T 95T 96S 97S 98S 99T 100S 101T 102T103S 104T105T 106T107T 109T110S 111S
M 112T 113S 114T 115T 116T 117T 118S 119T 120T 121T 122S 123T 124S 125S 126S 127T 128T 129T
1500bp
1030bp
1500bp
1030bp
M 130S 132S 133T 134S 135T 136S 137T 138S 139T 140T 141S 142S 143S 144T 145S 146T 147T 148T
112
1500bp
1030bp
M 149S 150T 151T 152S 153T 154T 155T 156S 157T 158T 159T 160T 161T 162T 163S 164T 165T 166T
Fig 4.11 TaCypB7A marker assay in the progeny from Tasman x Sunco. Data were generated from 161 DH lines and no PCR product in line (#51), for the TaCypB7A PCR-AFLP marker by primer pairs (CYPB-A1/CYPBR-A1). S indicates the phenotype being the same as Sunco. T indicates the phenotype being the same as Tasman.
The TaCypB7B marker was amplified from gDNA of the 162 DH lines of Tasman x
Sunco as first round PCR with WC1/CYPBR5, second round with CYPBF4/ CYPBR-
B1, and digestion of all second round products with HpyCH4IV (Fig 4.12). Seventy-
three lines out of 162 had the RFLP phenotype of Tasman, and 89 that of Sunco.
M 1T 2T 3S 4S 5T 6T 7S 8T 9T 10T 11T 12T 13S 14T 15S 16S 17T 18S
800bp 500bp
100bp
M 19S 20T 21S 22S 23T 24S 25S 26T 27S 28T 29T 30T 31T 32T 33S 34S 35T 36S
800bp
500bp
100bp
113
M 37S 38S 39S 40S 41S 42T 43T 44T 45S 46S 47T 48S 49S 50S 51S 52S 53T 54T
800bp
500bp
100bp M 55S 56T 57S 58T 59T 61T 62S 63T 64S 65S 66S 67T 68S 69S 70T 72S 74S
800bp
500bp
100bp
M 75S 76T 77T 78S 79S 80S 81S 82S 83T 84T 85S 86S 87S 88S 89S 90S 91S 92T
800bp
500bp
100bp
800bp
500bp
100bp
M 93S 94S 95S 96S 97S 98T 99T 100T 101S 102S 103S 104S 105S 106T 107T 109S 110T 111T
114
M 112S 113S 114S 115T 116T 117S 118T 119S 120T 121T 122T 123S 124T 125S 126S 127S 128T 129T
800bp
500bp
100bp
800bp
500bp
100bp
M 130T 132S 133S 134T 135S 136T 137S 138T 139T 140T141S 142T 143S 144S 145S 146S 147T 148T
M 149S 150S 151T 152T 153S 154T 155T 156T 157T 158S 159T 160T 161S 162S 163T 164T 165T 166T
800bp
500bp
100bp Fig 4.12 TaCypB7B marker assay in the progeny from Tasman x Sunco. Data was generated from 162 DH lines for the TaCypB7B marker, by first round PCR with WC1/CYPBR5, second round with CYPBF4/CYPBBR-B1, followed by digestion of the second round products with HpyCH4IV. S indicates the phenotype being the same as Sunco, T indicates the phenotype being the same as Tasman.
4.3.9 Linkage mapping of TaCypB7A and TaCypB7B
Analysis of the above data for TaCypB7A with the Map Manager software package
QTXb20 could not integrate this marker into the current genetic maps (data not shown)
due to limited markers in this area. In combination with location of bin mapped marker
(TaCypB7A: 7AL: 1-0.39-0.71) (Section 4.3.1), TaCypB7A is integrated in the reported
linkage map from Sunco x Tasman (Chalmers et al., 2001) (Fig 4.14A). This marker is
115
in the area (unbanded region 4) with no markers on this reported linkage map. The data
generated by Map Manager software package QTXb20 for TaCypB7B allowed its
integration between the markers wmc76 and wmc364 into the genetic map from the
Sunco x Tasman cross (Fig 4.13A) and on the same arm as gene Stb8 for resistance to
Septoris tritici blotch (Zwart et al., 2010) (Fig 4.13B). The TaCypB7B marker was
aligned into the comparative linkage map generated from a number of crosses including
Synthetic x Opata, Arina x Forno, CD87 x Katepwa, Cranbrook x Halberd, Egret x
Sunstar, and Sunco x Tasman (http://gramene.org/db/cmap/viewer, last accessed
07/2009) and found to be on the same arm as Stb8 (data not shown), as indicated in our
mapping (Fig. 4.13A), By combination of the Map Manager data with that from bin-
mapped marker (TaCypB7B: 7BL7-0.63-0.78) (Section 4.3.1), this marker was also
integrated in the reported linkage map from Sunco x Tasman (Chalmers et al., 2001)
(Fig 4.14B). The results showed that TaCypB7B was between markers (P40/M36-B and
xwmc364) on the linkage map of chromosome 7B (Figs 4.13A, 4.14B).
116
36-B
Fig 4.13 Linkage map of chromosome 7B generated from Sunco x Tasman (A) and comparative linkage map of chromosome 7B in T. aestivum (B). PCR-RFLP markers for TaCypB7B are highlighted (A) or shown in box between markers (Xwmc76 and Xwmc 364a) (B). Distances (in cM) and marker names are shown on the right and left of the map, respectively. Green indicates the AFLP markers and gene (B). Black indicates the RFLP, SSR markers (B).
117
1
123
4
5
6
7
TaCypB7A
A
118
P41/ M39-5, qwm400
Xwmc076
XsunM16
Xstm337b, gwm573
Xwg180b
P45/M32-2
P40/M 36-8
TaCypB7B
Xwmc402, Xwmc364
Stn337b
P39/M78
P42/M78
wmc273
gwm344
gwm577
ksuE19
rds481
B x Tasman
1 2
3
4
56
1
2
1
2
3
45
6
Fig 4.14 Integration of TaCYPB7A and TaCYPB7B markers in the linkage maps from Sunco x Tasman (Chalmers et al., 2001) based on the bin mapped markers indicated in Section 4.3.1 (Fig 4.5) and data analysis by Map Manager software package QTXb20 (Fig 4.13A). PCR-RFLP markers for TaCypB7A and TaCYPB7B are highlighted. Distances (in cM) and marker names are shown on the right and left of the map, respectively. Gray lines indicate the corresponding positions on chromosome 7A and 7B in three parental crosses.
119
4.3.10 Identification of QTLs on on chromosome arms of rice and wheat that
contain CypB genes
The QTLs on rice chromosome 6 in the general area of the CypB gene were identified
by browsing the Oryza sativa ssp. japonica Group genome database
(http://gramene.org) and looking up the chromosome location (29972988-29975061 bp)
corresponding to the putative rice CypB and looking up the QTLs in ContigView
(Gramene release V27 database). Fifty-five QTL accession IDs were identified, falling
in six categories: abiotic stress, anatomy, biochemical, quality, vigor and yield (Tables
4.2, 4.3). Three of these relate to grain quality i.e., AQFA001 (amylose content),
AQB008 (consistency viscosity) and AQCD004 (alkali digestion). Interestingly, the
consistency viscosity QTL (AQB008) (8.9cM) centred on a 1.3Mb region which
contained the CypB gene.
120
Table 4.2 QTLs in the general area of the rice CypB locus on chromosome 6
QTL Category
QTL Trait Name and Accession IDa
Genomic assembly
position (bp)b
Genetic position (cM)
Abiotic stress
iron sensitivity (AQEK008, AQEK006, AQEK012, AQEK004)
26,161,226-30,823,968
73.5-92.6
Anatomy
large vascular bundle number (AQBJ013, AQBJ004, AQBJ011)
28,638,436-30,823,968 84.4-92.6
panicle length (AQDY087, AQDY09, AQDY096, AQCB022, AQDY098, AQDY093, AQDY089)
26,161,226-30,823,968 71.1-87.9
leaf width (AQFW224) 29,027,995-30,945,466 227.1-234.3
internode length (CQX23), culm length (CQX9), panicle length (CQX14)
24,916,395-30,945,789 64.4-91.6
root branching (AQHJ008) 28,532,453-30,328,051 74.6-113.3
Biochemical
chlorophyll content (CQE47) 27,612,443-31,173,447 107.3-124.4
alpha amylase activity (AQAC035) 29,027,995-30,823,968 171-196
allelopathic effect (CQAD4) 28,695,316-30,945,789 116.7-135.6
blast disease resistance (AQAQ013, AQAQ004, AQAQ021)
29,027,995-30,945,466 223.7-234.3
white-backed planthopper resistance (AQBG012)
28,638,436-30,823,968 84.4-92.6
Quality amylose content (AQFA001) 29,027,995-30,447,453 90-128.2
consistency viscosity (AQB008) 28,431,560-30,328,051 133.5-143.7
alkali digestion (AQCD004) 27,252,300-30,265,590 0 -25.6
Vigor
plant height (AQBZ012), root dry weight (AQGI060, AQGI059)
29,027,995-30,823,968 171-196
tiller number (AQDY104, AQDY105) 26,161,226-30,823,968 60.6-87.9
root dry weight (AQHJ002) 28,532,453-30,328,051 74.6-113.3
ratooning ability (AQDK004), root to shoot ratio (AQBY008)
29,027,995-30,796,997 138.8-152.2
Yield
spikelet number (AQBK038) 29,539,579-30,823,968 85.1-85.1
spikelet number (AQCU041, AQCU243, AQFW114)
29,027,995-30,945,466 227.1-234.3
spikelet number (CQC5, AQBK006) 17,054,655-30,823,968 39.8-48.1
panicle number (AQDY103) 26,161,226-30,823,968 59.8-87.9
biomass yield (AQGI117, AQGI181), large vascular bundle number to spikelet number ratio (AQAI065), grain yield (AQCY003), 1000-seed weight (AQEY020), biomass yield (AQGI122), grain number (AQCQ013)
29,027,995-30,823,968
171-196, 148-162.7
yield (CQAS78) 29,502,932-30,796,997 132.9-142.3
panicle number (AQDY102) 21,731,316-30,823,968 49.7-87.9
seed number (AQBK042), seed set percent (AQBK045)
30,823,968- 3,168,374 17.7
aThe QTL accession ID in the list of QTLs, and the map set with associated markers close to
the CypB locus are given in parentheses. bGenomic assembly position (bp) in Gramene
Annotated Nipponbare Sequence 2006. The QTL related to quality are shaded and the details of the markers are listed in Table 4.3.
121
Table 4.3 Markers of QTLs controlling grain or protein quality on rice chromosome 6, in the general area of the CypB gene
Trait name and map seta Associated
Markersa
position in Gramene
sequence assembly (bp)b
Genetic position
(cM)b Amylose content (Cornell V20A/glab QTL 2004)
CDO78 N/A 90
RG653 29027995-29028429 109.2 RZ884 30446330-30447453 128.2
Consistency viscosity (Cornell IR64/Azu DH QTL 2001)
RG716 N/A 79.7
RG653 29027995-29028429 133.5 RM400 28431560-28431939 134.5
RM439 29624819-29625195 139.9
RM461 30113540 -30113752 139.9
RM412 30327854 -30328051 142.4
RM30 27252300-27252381 125.4 RM103 30889151-30889486 146.7
RM141 N/A 146.7
OSR21 N/A 146.7 Alkali digestion (CNYU Bal/Nan//Bal BC1 QTL 2001)
RM30 27252300-27252381 0 RM176 30265439-30265590 25.6
aThe trait name, map set, and list of associated marks were accessed from QTL database in website (http://gramene.org). The name of the relevant map set is given in parentheses. bPosition in Gramene sequence assembly and genetic position on the corresponding map set were accessed from Marker database on the same website.
QTLs on wheat chromosome 7 were identified by searching the integrated wheat
science database (http://www.shigen.nig.ac.jp, accessed 05/2008). Thirty-five QTLs
relating to agronomic and quality traits were present on group 7 chromosomes (Table
4.4, 4.5). Of these, QTLs related to flour, semolina and pasta colour were located on
chromosome 7 group, one for starch viscosity was on 7B between the markers
Xgwm344-7B (104.5cM) and Xwg420-7B (100.83cM) and 7A close to Wx-A (Batey et
al., 2002), flour and grain protein content on 7B close to Xwmc662 (Huang, et al.
2006), on 7A close to Xwmc65 and 7D close to between markers (Xcfd31–Xgwm44)
(Kuchel et al., 2006), grain protein content close to gwm263 on 7B and gwm332 on 7A
(Peleg et al., 2009), to wmc276-barc50/wmc311-Ta3 on 7B and wmc506-cfd66 on 7D
(Li et al., 2009), to Xgwm577 on 7B (Blanco et al., 2006).
122
Table 4.4 Wheat QTLs relating to agronomic and quality traits on chromosome 7* Classes Trait
Grain Quality Parameters
Flour, semolina and pasta color
Starch characteristics Loaf volume
Proteins Grain protein content Endosperm storage proteins (glutenins) Enzyme Inhibitors (subtilisin inhibition)
Yield and physiology Restorers for cytoplasmic male sterility Grain weight
Flowering time
Dormancy (seed) Purple grain/pericarp
Defence from diseases and toxins
Reaction to Diuraphis noxia (Mordvilko) Resistance to disease caused by Fusarium head scab, scab Resistance to Blumeria graminis
Reaction to crown rot caused by Fusarium pseudograminearum, F. culmorum and other Fusarium species
Reaction to Schizaphis graminum Rond. Resistance to Phaeosphaeria nodorum (E. Muller).
Sensitivity to SNB toxin Reaction to Puccinia striiformis Westend (Strip rust QTLs). Resistance to tan spot
Reaction to Black-Point of Grain Resistance to disease caused by Mycosphaerella graminicola (Fuckel) Schroeter
Vigor Growth rate and early vigor *The QTLs in wheat on chromosome 7 were accessed from integrated wheat science database
(http://www.shigen.nig.ac.jp, accessed 05/2008).
123
Table 4.5 Summary of wheat QTLs for agronomic and quality traits and their closest markers on chromosome 7
Trait Chr Closest or interval Markers Wheat varieties Reference
Septoria tritici blotch (Stb)
7D wPt-3727; Xwmc405b DHLs from F1 of CPI133872/Janz
Zwart et al., 2010 Stripe rust 7D wPt-2863 (8.8) wPt-3727
Red coleoptile
7A Xwmc283; wPt-3883/wPt-4877; wPt-8897, wPt-4744
7D Xwmc405b; Xwmc405a; Xpsp3123 Grain yield 7A Xwmc83 DHLs from F1 of AC
Karma/87E03-S2B1 Huang et al., 2006 7B Xgwm537
Plant height 7B Xgwm537
Grain protein 7B Xwmc662
Flour protein 7B Xwmc662
Flour protein 7A Xwmc65 DHLs derived from F1 of Trident/ Molineux
Kuchel et al., 2006 7D Xcfd31–Xgwm44
Flour color 7B Xgwm273–Xgwm146
Milling 7D Xgwm130 DHLs derived from F1 of RL4452/‘AC Domain
McCartney et al., 2006 Mixograp 7D Xgwm130–Xwmc405
7B Xgwm400–Xwmc182 Starch 7A Xwmc139, Xwmc422
7D Xgwm130–Xwmc405, Xwmc702 Noodle colour 7A Xwmc633–Xwmc809 Baking 7A Xbarc70-Xwmc168 Resistance to Fusarium head blight
7A Xgwm276-Xgwm282; DHLs derived from F1 of Wangshuibai/Alondra
Jia et al., 2005 7B Xgwm297-Xgwm644; Xgwm146-
Xgwm611 Grain yield 7AL Xpsp3094.1-Xm68p78.6 DHLs from F1 of Chinese Spring × SQ1
Quarrie et al., 2004 7BL Xm43p78.14-Xm86p65.0
7BS Xm59p78.7
Starch viscosity 7A Wx-A1 DHLs from F1 of CD87/Katepwa Batey et al., 2002 7B Xgwm344-Xwg420 DHLs derived from F1 of
Cranbrook / Halberd Grain hardness 7B wmc517-wmc311 RILs from Neixiang 188/ Yanzhan 1
Li et al., 2009
Grain protein 7B wmc276-barc50/wmc311-Ta3
7D wmc506-cfd66 Wet gluten content 7B wmc276-barc50/wmc311-
7A wmc506-cfd66 Water absorption 7D wmc506-cfd66
Grain protein 7A gwm332 RILs from durum wheat/wild emmer wheat
Peleg et al., 2009 7B gwm263
Grain zinc 7A wPt-9555
7B gwm983
Grain iron 7A wPt-9555
7B gwm400
Grain copper 7A wPt-7053
7B gwm46
Grain manganese 7B gwm400 Grain calcium 7B gwm263
Grain magnesium 7A gwm871a
7B wPt-8417
Grain Potassium 7B gwm537
Grain Phosphorus 7A wPt-7053
Grain sulfur 7A gwm1083
7B wPt-3730
Loaf volume 7A cfa2049, bcd1930 RILs from Renan and Récital Groos et al., 2007 7B gpw1045, gwm577
S. nodorum resistance
7A Xglk165- pwir232a RILs from Forno and Oberkulmer Aguilar et al., 2005 7B Xpsr952- Xgwm46
Powdery mildew 7BL Xpsr593c-Xpsr129c RILs from Forno and Oberkulmer Keller et al., 1999 Grain protein 7B Xgwm577 BILs derived from durum wheat/
dicoccoides Blanco et al., 2006 AB: advance backcross; BIL: backcross inbred line; DHL: double hybridization lines; RIL
(recombinant inbred lines). QTLs related to wheat grain or proteinquality traits on group of chromosome 7 are shaded.
124
4.4 Discussion
4.4.1 Data on physical mapping of wheat in rice database indicates CypB genes
may be duplicates on different chromosomes
The wheat orthologues (wheat bin mapped markers) were identified at the putative rice
CypB locus LOC_Os06g49480. Three ESTs in wheat corresponding to the rice CypB
locus were mapped on chromosomes 1 and 7 and all three ESTs showed very high
identity (> 97%) with exon contig of TaCypB-B. This observation indicates that CypB
genes may be located on chromosome 1 and/or 7suggesting possible recent duplications.
4.4.2 Development of molecular markers for physical mapping prior to designing
of allele specific primers
With no information on allele specific primers at this stage, a PCR based AFLP+RFLP
assay was developed for physical mapping of the genes onto specific chromosomes.
The SNPs in the three CypB genes (Chapter 3) provided the basis for exploiting these
polymorphisms. As some of SNPs were expected to alter restriction sites, RFLP was
applied to conveniently identify the polymorphisms between homeoalleles. The success
of this approach in mapping shows this may be a most useful tool for mapping of other
genes too. All CypB genes mapped to chromosome 7AL, 7BL, and 7DL through
application of PCR-based analysis of polymorphisms to the nullisomic-tetrasomic lines
and deletion lines; however, the wheat bin mapped-markers data (from rice genome)
suggested CypB could be on wheat chromosomes 1 as well. This may be due to the
primers being too specific for amplifying a CypB gene on chromosome 1, if it is present
but divergent. The copy number of CypB genes thus needs to be addressed by methods
such as southern blotting, with the isolated CypB cDNA clones (section 3.3.6) as
probes. To author’s knowledge, this is the first report of physical mapping of CypB
genes in wheat. The data provide the basis for analysis of expression of the three
individual homeoalleles.
125
4.4.3 Genetic mapping of TaCypB7A
The presence of SNPs in the genomic sequences of three CypB genes allows the design
of allele specific primer pairs for amplification genes from A, B and D genomes of
common wheat (Table 4.1). Use of these primer pairs in ten mapping cultivars showed
an AFLP polymorphism between the parental pair Tasman x Sunco. The genotyping
data of the TaCypB7A marker in 161 DH progeny lines of this cross were analysed for
integrating this gene on the linkage map of chromosome 7A; however, the gene could
not be integrated by the Map Manager software package QTXb20, possibly due to
limited markers. TaCypB7A was integrated in the reported linkage map from Sunco x
Tasman (Chalmers et al., 2001) (Fig 4.14A) with combination of its bin mapped marker
location (7AL: 1-0.39-0.71) (Section 4.3.1). This marker is in the area with no markers
on this reported linkage map. Future efforts could concentrate on finding SNPs of
other parental pairs, e.g. Cranbrook x Halbred or CD87 x Katepwa.
4.4.4 Genetic mapping of TaCypB7B
No AFLP was found in the B genome, but one SNP (T-C) altered the HpyCH4IV site,
leading to RFLP polymorphism in the mapping cultivar crosses Tasman x Sunco, CD87
x Katepwa and Janz x Kukri. The genotyping of TaCypB7B marker in the 162 DH
progeny lines from Tasman x Sunco permitted its integration onto the genetic linkage
map of 7B and reported linkage map from Sunco x Tasman (Chalmers et al., 2001) (Fig
4.13A, 4.14B). This linkage map contains markers including AFLP, RFLP, SSR and
gene specific markers, and this marker is located between markers (P40/M36-B and
xwmc364). TaCypB7B appears close to gene Stb8 for resistance to septoria tritici
blotch (Zwart et al., 2010) on the linkage maps generated from a number of crosses
including Synthetic x Opata, Arina x Forno, CD87 x Katepwa, Cranbrook x Halberd,
Egret x Sunstar, and Sunco x Tasman (http://gramene.org/db/cmap/viewer). The gene
may be useful as a marker by identifying any QTLs close to or co-localised with it.
4.4.5 Genetic mapping of TaCypB7D
Neither AFLP nor SNPs were found in the partial TaCypB7D in ten mapping lines.
This is in agreement with reports of lower levels of polymorphisms within D genome of
T. aestivum (Langridge et al., 2001). Further efforts to develop a molecular marker for
TaCypB7D gene may require for analysis of the 5’-end of this gene.
126
4.4.6 QTLs in the chromosome regions of the cyclophilin B genes in rice and wheat
Several QTLs in rice have been identified on the region 17,054,655-3,168,374 bp on
chromosome 6 (Table 4.2). The QTLs encoding blast resistance and white-backed plant
hopper resistance are in the region 28,638,436-30,945,466 bp. Several QTLs resistant
to diseases are located on chromosome 7 in wheat (Tables 4.4, 4.5). The wheat Cyp-
like protein homologous to Arabidopsis AtCYP21-4 (Romano et al., 2004a) has been
reported to be involved in resistance to one of the most important diseases (yellow rust)
(Bozkurt et al., 2008). The QTLs related the traits of plant height and root dry weight
are at the 29,027,995-30,823,968 bp region. The ER-localised AtCYP20-1 in
Arabidopsis has a role in regulating PP2A activity for auxin transport and growth
response pathways (Jackson and Soll, 1999). Thus it is reasonable to ask whether the
wheat CypB gene may also have functions in defence or plant growth; these will need to
be tested further.
Three quality QTLs, relating to amylose content, consistency viscosity and alkali
digestion, occur in the general area of the putative rice CypB (Table 4.3). The QTLs for
consistency viscosity are in the region (28,431,560-30,328,051 bp) on chromosome 6,
which contains the CypB gene. Most interestingly, the wheat QTL search also shows a
probable association with consistency viscosity. A number of QTLs relating to
agronomic and quality traits occur on wheat group 7 chromosomes. In particular, the
consistency viscosity QTL in rice centres on the chromosome 6 region containing the
CypB gene and the wheat QTL for starch viscosity is on 7BL, between the markers
Xgwm344-7B (104.5cM) and Xwg420-7B (100.83cM) (Batey et al., 2002). This
observation supports the reported synteny of a large section of wheat group 7
chromosomes to rice chromosome 6 (La Rota and Sorrells, 2004). The visco-elastic
properties of dough have long been known to be important to the quality of bread, pasta,
noodles and other foods (reviewed in Payne 1987; Shewry 2009). Any causative
association of the CypB genes with this trait, seen in both cereals, thus suggests could
play significant roles in influencing storage protein quality. This is a very important
outcome of this work and will need to be tested further by identification of genetic
variations or gene knock
127
Chapter 5
A COMPARATIVE PHYLOGENETIC AND MOLECULAR ANALYSIS OF THE
CYCLOPHILIN FAMILY IN RICE AND WHEAT
128
5. A comparative phylogenetic and molecular analysis of the
cyclophilin family in rice and wheat
5.1 Abstract
Cyclophilins are a group of peptidyl prolyl cis/trans isomerase enzymes that catalyse the
slow cis/trans isomerisation of the peptide bond preceding proline residues in the
nascent protein folding and refolding processes. A large cyclophilin (Cyp) gene family
in Arabidopsis has been reported, with twenty-nine members located in all cellular
compartments and performing numerous functions. Little is known about the Cyp
family in rice and wheat, except reported three members in rice, and two members in
wheat. The genomic loci encoding the Cyp gene family in Arabidopsis were used as
queries to search Rice Annotation Release 6 in rice and Plant Transcription Assembly
(TA) database in wheat. Thirty-three Cyps encoded by twenty-seven loci in rice and
twenty-two Cyps encoded by TAs in wheat were identified, located in five cellular
locations. The gene encoding Cyps in rice are located on 11 different chromosomes and
range from being intronless to having 14 introns. Three out of twenty-two of TAs
encoding Cyps in wheat mapped chromosomes 1, 3, and 7. In addition to TaCYP23-1
(cyclophiln B, chapter 3), two other potential ER localized cyclophilins (TaCYP24-1
and TaCYP26-2) were identified in this study. Three genes encoding ER-localised
Cyps were found to be expressed in different tissues and developing seeds by RT-PCR
analysis. The TA data of Cyps in wheat can be used to develop markers for mapping in
wheat.
129
5.2 Introduction
cyclophilins (Cyps) are a group of peptidyl prolyl cis/trans isomerase enzymes that
catalyse the otherwise-slow cis/trans isomerisation of the peptide bond preceding
proline residues during the folding and refolding of newly formed proteins (Galat, 1993;
Wang and Heitman, 2005) (sections 1.3, 1.4). Their activity is inhibited by the
immunosuppressive drug cyclosporin A (CsA) (Handschumacher et al., 1984).
Cyclophilins are ubiquitous and can be grouped based on their subcellular locations, i.e.
cytosol, endoplasmic reticulum (ER), nucleus, mitochondria, and chloroplast. Chapter 1
details the various functions of Cyps in humans and other organisms, in their PPIase
and/or chaperone roles. In brief, the cytosolic Cyps may play roles in the interaction
with infected species (Pelle et al., 2002), plant growth (Oh et al., 2006; kielbowicz-
Matuk et al., 2007), response to stress conditions (Godoy et al., 2007; Dubery, 2007),
and enhancing multiple abiotic stress tolerance (Kumari et al., 2009). A cytosolic multi-
domain Cyp containing tetratricopeptide repeats (TRP) is required for vegetative phase
change (Berardini et al., 2001). The 65kDa Cyp with U-box acts as a prolyl isomerase
and a chaperone for resolving protein aggregates (Wiborg et al., 2008). Cyp with WD
repeats act as a highly conserved histone remodeling factor involved in gene silencing
(Li et al., 2007). The ER-localised Cyps act as a transcriptional inducer (Rycyzyn and
Clevenger, 2002), play crucial roles in protecting cells against ER stress (Kim et al.,
2008), and regulate PP2A for auxin transport and growth response (Jackson and Soll,
1999). The chloroplast Cyps function in catalysing the correct folding and integration
of proteins in the thylakoid membrane (Edvardsson et al., 2003; Romano et al., 2004b)
and repairing photodamaged photosystem II (PSII) (Cai et al., 2008). A chloroplast Cyp
containing leucine zipper motif plays a critical role in the assembly and maintenance of
PSII supercomplexes (Fu et al., 2007; Sirpio et al., 2008). Nucleus localized Cyps with
an Arg/Lys domain play roles in cell cycle regulation (Dubourg et al., 2004) and pre-
mRNA processing (Lorkovi et al., 2004; Gullerova et al., 2007), while the
mitochondrial Cyps have roles in protection against apoptosis (Lin and Lechleiter,
2002).
The complexity of the Cyp gene family, consisting of 29 members in Arabidopsis (He et
al., 2004; Romano et al., 2004a) gives a clue to the potential diversity of Cyps in plants,
but little detail has been uncovered in other plants, especially cereals. Two cDNA
130
clones from rice encode two different Cyps (Cyp1 and Cyp2) with differential
expression in leaf and root, and the genomic Cyp2 is intronless (Buchholz et al., 1994).
One previously reported cytosolic member (CypA) from wheat genomic DNA is
intronless (Johnson and Bhave, 2004b). In the experiments described in chapters 3 and
4, an ER-localised CypB was isolated from wheat, compared to one identified from rice
genome, physically mapped to group 7 chromosomes, linkage-mapped, and shown to be
in the genetic region associated with certain grain protein quantitative traits.
In light of the diverse functions of human and other plant Cyps (see above and Chapter
1), it is essential to develop an understanding of this gene family and identify members
with potential contributions to traits such as grain quality, plant development and stress
response. The Cyp family has not been studied in plants other than Arabidopsis. This
chapter reports bioinformatics-based identification of the Cyp family in rice from Rice
Annotation Release 5, and in wheat from the Plant Transcription Assembly database.
Further, analysis of gene structures in rice, estimation of phylogenetic relationships of
Cyps in rice, wheat and Arabidopsis, and preliminary expression analysis of genes
encoding ER-localised Cyps in wheat were conducted.
131
5.3 Results
5.3.1 Identification of genomic sequences of Cyps genes in rice
The search for Cyp family members in rice (Oryza sativa ssp japonica cv. Nipponbare)
in the Rice Annotation Release 5 (and updated from Release 6)
(http://rice.plantbiology.msu.edu/osa1.shtml) using the reported Arabidopsis Cyp family
sequences as queries (Romano et al., 2004a) led to identification of 27 loci, resulting in
33 coding sequences (CDS; or exons contigues) due to some loci encoding different
splice forms (Table 5.1). The gene names were assigned based on the molecular weight
of putative proteins (introduced later). Twenty-two loci encoded single splice forms,
four (LOC_Os06g49480, LOC_Os07g37830, LOC_Os08g19610, LOC_Os08g44330)
potentially encoded two splice forms, and LOC_Os08g44520 presented three (Fig 5.1;
DNA alignments of alternative splice forms shown in Appendix V). Of these possible
alternative forms,
(i) OsCYP24-1 was smaller than OsCYP24-2 by 15 bp in exon I but larger than it by
15 bp in intron I.
(ii) OsCYP46 was split into two exons at the 5’-end by a 99 bp intron I, compared to
OsCYP50 containing 444 bp in exon I.
(iii) OsCYP40 had an extra exon II (212bp) and intron I (2716 bp), compared to the
intronless OsCYP28.
(iv) OsCYP73 had three extra exons (245 bp, 86 bp, 89 bp) and three extra introns (86
bp, 323 bp, 162 bp) at 5’-end, compared to OsCYP58.
(v) OsCYP18-2 was larger than OsCYP19-3 by 36 bp in intron III but smaller than it
by 36 bp in exon III.
(vi) OsCYP20 had extremely large intron I (1352 bp), compared to OsCYP18-2 and
OsCYP19-3 (120 bp).
5.3.2 Identification of expressed sequences of Cyp genes in rice
Thirty-one expressed Cyp sequences were also retrieved from the rice Transcript
Assembly (TA) database (see methods in section 2.19). Two sequences (OsCYP19-3,
OsCYP58) did not have a corresponding TA, suggesting these splice forms may not be
expressed, or at least not in the tissues that the ESTs were reported.
132
5.3.3 Predictions of intron/exons structures of rice Cyp genes
The genomic sequences from the 27 loci ranged from 420 bp (LOC_Os02g02090) to
8092 bp (LOC_Os02g10970) and the coding sequences (CDSs) (exon contigs) ranged
from 420 bp (OsCYP16) to 1956 bp (OsCYP73) (Table 5.1). The gene structures
generated by comparison of genomic DNA with corresponding CDS varied from
intronless (OsCYP16, OsCYP18-1, OsCYP18-3, OsCYP19-1, OsCYP19-2, OsCYP28) to
a 15-exon-14-intron structure (OsCYP63) (Fig 5.1) (discussed later).
133
Fig 5.1 Gene structures of Cyp gene family in rice Gene structures were generated based on comparison of genomic sequences with the corresponding CDSs downloaded from the Rice Annotation Release 6 (http://rice.plantbiology.msu.edu/osa1.shtml) with program ‘gene structure display server’ (http://gsds.cbi.pku.edu.cn). Blue lines indicate 5’ and 3’ extra regions compared to the CDSs. Green boxes indicate exons. Regions marked in red indicate the DNA sections encoding main domain in putative Cyp proteins.
134
Table 5.1: List of Rice Cyclophilin gene family*
Namea Locus ID
b
GS
c
(bp)
CDS
(bp)
Ex
on
cDNA/EST RFWHd Protein
(KDa/aa) Cellular location
e
OsCYP16 Os02g02090 420 420 1 BE229368 QIWY 16.2/139 Cytosol
OsCYP18-1 Os06g04000 934 483 1 AK105360 RFHY 17.5/160 Cytosol
OsCYP18-2 Os08g44520 3804 495 6 AK072675 RFSH 18.0/164 Cytosol
OsCYP18-3 Os02g02890 1078 519 1 AK061894 RFWH 18.3/172 Cytosol
OsCYP19-1 Os10g06630 1015 546 1 AK058898 RFWH 18.9/181 Cytosol
OsCYP19-2 Os09g39780 1101 540 1 AK103109 RFWH 19.2/179 Cytosol
OsCYP19-3 Os08g44520 3804 531 6 na RFSH 19.4/176 Cytosol
OsCYP20 Os08g44520 3804 537 6 CF957035 RFSH 19.8/178 Cytosol OsCYP22-1 Os03g59700 3099 615 7 AK071076 RFWH 21.8/204 Cytosol
OsCYP22-2 Os06g49470 2197 627 7 CI598173 RFRH 22.1/208 ER OsCYP23 Os09g36670 2623 648 7 AK061004 RFWH 23.4/215 ER
OsCYP24-1 Os06g49480 2478 663 7 AK072490 RFWH 23.5/220 ER
OsCYP24-2 Os06g49480 2478 678 7 AK060865 RFWH 24.0/225 ER OsCYP25 Os01g18210 3133 717 6 CI392401 RFWH 25.4/238 CP OsCYP26-1 Os11g38990 5003 708 7 AK068821 RFHY 25.8/235 ER OsCYP26-2 Os07g29390 5767 708 7 AK066346 HFDL 26.4/235 M OsCYP27 Os05g01270 2866 753 7 AK061557 RFWH 26.6/250 CP
OsCYP28 Os08g19610 4898 777 1 AK121304 KQEN 28.2/258 CP OsCYP31 Os01g02080 1367 915 2 AK065672 KYEA 31.4/304 CP OsCYP40 Os08g19610 4898 984 2 AK069211 KQEN 35.9/327 CP OsCYP43 Os06g11320 5858 1191 8 AK068112 RFHH 43.2/396 Cytosol OsCYP44 Os02g52360 4560 1212 8 AK070404 RFHH 44.0/403 Cytosol
OsCYP46 Os07g37830 5593 1299 12 AK071983 QDFF 46.1/432 CP
OsCYP47 Os08g29370 3918 1284 7 AK058264 RKIY 46.6/427 CP
OsCYP49 Os02g10970 8092 1311 10 AK068982 RFRS 49/436 Nucleus
OsCYP50 Os07g37830 5593 1398 11 AK107481 QDFF 49.8/465 CP
OsCYP51 Os03g10400 4020 1389 7 AK102337 RFHH 50.7/462 Cytosol
OsCYP57 Os01g40050 4816 1500 10 AK069484 RFWN 56.8/499 Cytosol
OsCYP58 Os08g44330 5595 1542 10 na RFWH 58.3/513 Cytosol
OsCYP63 Os06g45900 8079 1647 15 CI140095 KFYH 63.2/548 Nucleus
OsCYP65 Os06g45910 6072 1695 14 AK059079 KFYH 65.0/564 Nucleus
OsCYP70 Os07g08190 6218 1899 13 AK059946 RFHH 70.0/632 Nucleus
OsCYP73 Os08g44330 5595 1956 13 AK111654 RFWH 72.9/651 Cytosol
* The Cyp family was identified originally in Rice Annotation Release 5, and updated from Rice Annotation Release 6) (http://rice.plantbiology.msu.edu/, last accessed 02/2009).
a Cyps were
named based on estimated molecular weight of putative proteins; names in bold refer to sequences reported by other names, i.e. OsCYP18-3 and OsCYP19-2 reported as Cyp2 and Cyp1 (Buchhoz et al., 1994); OsCYP24-1 designated as CypB (Chapter 3).
bLoci in bold encode
two or three putative Cyps. cGS: genomic sequences.
dFour residues essential for PPIase
activity and CsA binding. eCellular locations predicated ‘PSORT Prediction’ (http://psort.ims.u-
tokyo.ac.jp), and ‘TargetP 1.1 Server’ (http://www.cbs.dtu.dk/services/TargetP); M: mitochondria; CP: chloroplast.
5.3.4 Grouping of putative Cyp proteins in rice
The sizes of putative proteins ranged from 16.2 kDa (139aa) of OsCYP16 to 72.9 kDa
(651aa) of OsCYP73. Cellular locations were predicted by ‘WoLF PSORT Prediction’
and ‘TargetP 1.1 Server’ (section 2.22) as well as comparisons to those in Arabidopsis
135
(He et al., 2004; Romano et al., 2004a), and based on the results, the rice Cyps were
grouped into cytosolic, ER, nuclear, mitochondrial and chloroplast isoforms (Fig 5.2).
The Cyps in wheat were identified and sequences of all three species (rice, wheat,
Arabidopsis) were compared (details presented later). The key characteristics of each
group in rice were described below.
nucleusAtCYP59 OsCYP49 TaCYP70AtCYP63 OsCYP63 AtCYP95 OsCYP65
OsCYP70
Cyclophilins in rice, wheat, and
Arabidopsis
ER
AtCYP19-4 OsCYP22-2 TaCYP23-1 AtCYP20-1 OsCYP23 TaCYP24-1AtCYP21-1 OsCYP24-1 TaCYP26-2AtCYP21-2 OsCYP24-2 AtCYP23-1 OsCYP26-1
Mitochondria AtCYP21-3 OsCYP26-2 TaCYP16-1 AtCYP21-4 TaCYP23-2
TaCYP26-4
ChloroplastAtCYP20-2 OsCYP25 TaCYP26-1 AtCYP20-3 OsCYP27 TaCYP26-3 AtCYP26-2 OsCYP28 TaCYP32 AtCYP28 OsCYP31 TaCYP36 AtCYP37 OsCYP40 TaCYP46-2 AtCYP38 OsCYP46
OsCYP47OsCYP50
CytosolAtCYP18-1 OsCYP16 TaCYP16-2 AtCYP18-2 OsCYP18-1 TaCYP17 AtCYP18-3 OsCYP18-2 TaCYP18-1 AtCYP18-4 OsCYP18-3 TaCYP18-2 AtCYP19-1 OsCYP19-1 TaCYP19 AtCYP19-2 OsCYP19-2 TaCYP21 AtCYP19-3 OsCYP19-3 TaCYP24-2 AtCYP22-1 OsCYP20 TaCYP31-1AtCYP26-1 OsCYP22-1 TaCYP31-2AtCYP40 OsCYP43 TaCYP46-1 AtCYP57 OsCYP44 AtCYP65 OsCYP51 AtCYP71 OsCYP57
OsCYP58 OsCYP73
Fig 5.2 Predicted cellular locations of cyclophilins in wheat and rice compared to those reported in Arabidopsis (He et al., 2004; Romano et al., 2004a; 2005). Cellular locations of Cyps were predicated by ‘PSORT Prediction’ (http://psort.ims.u-tokyo.ac.jp) and ‘TargetP 1.1 Server’ (http://www.cbs.dtu.dk/services/TargetP).
Fifteen members were predicted to be located in cytosol, encoded by eleven loci. The
estimated lengths/molecular weights of the putative full-length proteins ranged from
139 aa/16.2 kDa (OsCYP16) to 651 aa/72.9 kDa (OsCYP73) (Table 5.1). OsCYP18-3
and OsCYP19-2 were earlier reported as Cyp2 and Cyp1, respectively (Buchholz et al.,
1994). Based on alignment with the reported human CypA (hCyP18-a, NP_066953)
and orthologues in Arabidopsis (Romano et al., 2004a) (introduced later), the structures
of Cyps in this group are shown in Fig 5.3. Nine members out of fifteen (OsCYP16,
136
OsCYP18-1, OsCYP18-2, OsCYP18-3, OsCYP19-1, OsCYP19-2, OsCYP19-3,
OsCYP20, OsCYP22-1) contained only the main domain for PPIase activity (Galat,
1999). OsCYP51 contained the extra section at both the N- and C-termini with no
functions known. Five (OsCYP43, OsCYP44, OsCYP57, OsCYP58, OsCYP73) were
multi-domain, of which OsCYP43 and OsCYP44 contained the main domain at the N-
terminus and the tetratricopeptide repeat (TPR) at the C-terminus shown to be required
for association with the Hsp90 chaperone complex (Duina et al., 1996). OsCYP58 and
OsCYP73 contained the WD40 repeat for protein-protein interactions (reviewed in He
et al., 2004). OsCYP57 had an Arg/Lys rich domain at the C-terminus with nucleus
localisation signals (NLS), characteristic of RNA-interacting proteins (Fu, 1995).
Five members were predicted to be located in ER, encoded by four loci. The putative
full-length proteins ranged from 208 aa/22.1 kDa (OsCYP22-2) to 235 aa/25.8 kDa
(OsCYP26-1). OsCYP24-1 is the CypB analysed in Chapter 3. All members had ER-
targeting signals at N-terminus (Price et al., 1991) and were predicted to have a SP
(secretory pathway signal peptide) by ‘TargetP 1.1 Server’ (Fig 5.3). The SP cleavage
site was predicted to be between residues A28 and V29 (OsCYP22-2), A20 and A21
(OsCYP23), A39 and K40 (OsCYP24-1), A37 and Q38 (OsCYP24-2) and G27 and
A28 (OsCYP26-1). Based on this, the mature proteins were estimated to be 19.2-23.3
kDa.
Eight members were predicted to locate in the chloroplast, encoded by six loci. The
putative full length of proteins ranged from 238 aa/25.4 kDa (OsCYP25) to 465 aa/49.8
kDa (OsCYP50) (Table 5.1). The putative N-terminal bipartite chloroplast/thylakoid
(C/T) targeting signals for translocation into thylakoid lumen, predicted by the ‘TargetP
1.1 Server’, existed in all putative homologues (Fig 5.3). OsCYP47 contained the
seven-residue repeat regions of a putative leucine-zipper motif between the C/T
targeting signals and main domain (Fulgosi et al., 1998).
One member was predicted to be mitochondrial, the full-length protein being 235
aa/26.4 kDa (OsCYP26-2). It contained the mitochondrial targeting signal (MTS) at N-
terminus and the main domain at the C-terminus (Fig 5.3).
137
Four members, encoded by four loci, were predicted to be nuclear. The lengths/
molecular weights ranged from 436 aa/49.0 kDa (OsCYP49) to 632 aa/70.0 kDa
(OsCYP70) (Table 5.1). All members contained the Arg/Lys domains at the C-terminus
with SR rich motif within this domain, considered as the nucleus localisation signals
(NLS), characteristic of RNA-interacting proteins (Fu, 1995). OsCYP63 and OsCYP65
contained unique RNA recognition motif (RRM) involved in binding to RNA (Birney et
al., 1993) and Zinc finger (Z/F) motif for mediating DNA binding, protein-protein and
protein-lipid interaction (reviewed in He et al., 2004) (Fig 5.3).
139OsCYP16
160OsCYP18-1
164OsCYP18-2
172OsCYP18-3
181OsCYP19-1
179OsCYP19-2
176OsCYP19-3
17 178OsCYP20
33 204OsCYP22-1
36 208OsCYP22-2
34 215OsCYP23
ER47 220
OsCYP24-1
52 225ER OsCYP24-2
OsCYP2569 238
33 214OsCYP26-1
MTS
79 250
OsCYP26-2
OsCYP27
83 304
106 258OsCYP28
OsCYP31
70 235
C/T
106 327C/T OsCYP40
22 208OsCYP43
29 215OsCYP44
238 432OsCYP46
226 427OsCYP47
OsCYP491 190
271 465OsCYP50
219 387OsCYP51
1 181OsCYP57
353 513OsCYP58
1 205OsCYP63
1 175OsCYP65
1 187OsCYP70
491 651 OsCYP73
ER
C/T
ER
C/T
C/T
L/Z
101 160C/T
83
116
C/T
C/T
TPR TPR TPR
TPR TPR TPR
286 349
293 356
272/347
RRM
367/380
242/320 340/353
RRMZ/F
R/K
R/K
WD WD WD86 240
WD WD3 122
R/K
199 499
224 632
R/K
421 548
395 564
376/436
ER
R/K
Fig 5.3 Structure of putative Cyp proteins in rice. Grey box indicates main domain for PPIase catalysis; ER: ER-targeting signal; C/T: chloroplast/ thylakoid lumen targeting signal; MTS: mitochondrial targeting signal; TPR: tetratricopeptide repeat; R/K: Arg/Lys rich domain; dark bars in the R/K domain indicate NLS (nuclear localisation
signal). WD: WD40 repeat; RRM: RNA recognition motif; L/Z: Leu zipper; Z/F: Zinc-finger.
138
5.3.5 Analysis of the rice Cyp gene structures
The lengths of individual exons varied from 24 to 795 bp (Table 5.2) and those of
introns, from 0 to 2716 bp (Fig 5.1; Appendix VI). All exhibited GT_AG at the
putative intron/exon splice junctions, except the intron I of OsCYP25 showing AG_GC
(data not shown; Appendix V). Genes encoding single domain cytosolic Cyps were
found to be intronless (OsCYP16, OsCYP18-1, OsCYP18-3, OsCYP19-1, OsCYP19-2)
or had five to six exons (OsCYP18-2, OsCYP19-3, OsCYP20). Genes encoding
OsCYP43 and OsCYP44 had the same 8-exon structure, the sizes of exons II, V, VI
VII, VIII being conserved (Table 5.2), the main domain encoded in exons I and II (Fig
5.2), and TPR domain mainly in exons V-VIII. The identities of CDS and putative
amino acid sequences of OsCYP43 and OsCYP44 were 81.2% and 82.6%, respectively.
The results suggest that their loci may be duplicate genes, dispersed on these two
chromosomes.
Genes encoding the putative ER Cyps had the same 6-exon structure. The sizes of
exons II, IV, V VI were conserved in OsCYP22-2, OsCYP23, OsCYP24-1, OsCYP24-2
(Table 5.2). The main domain was encoded by exons II-VI (Fig 5.2). The identities of
CDS and putative amino acid sequences of OsCYP24-1 and OsCYP24-2, possibly
different splice forms of LOC_Os06g49480, were both 97.8%. Further, the CDS of
OsCYP24-1 had high identities to those of OsCYP22-2 (73.5%) and OsCYP23 (57%),
and the respective protein identities were also 69.5 or 52.2%. These results suggest
that the loci LOC_Os06g49470, LOC_Os06g49480 and LOC_Os09g36670 may be
duplicates dispersed on two chromosomes, of which, LOC_Os06g49470 (29970113–
29971975 bp) and LOC_Os06g49480 (29972988–29975061 bp) are separated by only
1013 bp.
Genes encoding the two putative nuclear Cyps had conserved sizes of exons V-VIII of
OsCYP63 to exons IV-VII of OsCYP65. The identities of CDS and putative amino acid
sequences of OsCYP63 and OsCYP65 were 76.8% and 69.3%, respectively.
LOC_Os06g45900 (27781934–27790012 bp) and LOC_Os06g45910 (27791762–
27797300 bp) may thus be duplicate genes, separated by only 1751 bp.
139
Table 5.2 Summary of exon sizes (bp) of Cyp genes in rice*
Exon I
Exon II
Exon III
Exon IV
Exon V
Exon VI
Exon VII
Exon VIII
Exon IX
Exon X
Exon XI
Exon XII
Exon XIII
Exon XIV
Exon XV
OsCYP18-2 72 96 62 134 85 46
OsCYP19-3 72 96 98 134 85 46
OsCYP20 114 96 62 134 85 46
OsCYP22-1 209 24 88 93 33 57
OsCYP22-2 81 79 48 24 183 76 136
OsCYP23 78 75 48 24 183 76 163
OsCYP24-1 114 79 48 24 183 76 139
OsCYP24-2 129 79 48 24 183 76 139
OsCYP25 184 147 89 55 86 156
OsCYP26-1 138 114 149 73 90 40 90
OsCYP26-2 152 40 51 86 138 148 93
OsCYP27 30 189 151 89 55 68 153
OsCYP31 796 118
OsCYP40 772 212
OsCYP43 237 501 99 46 105 76 38 82
OsCYP44 258 501 186 53 105 76 38 82
OsCYP46 165 180 90 95 108 50 103 54 156 123 50 124
OsCYP47 117 288 200 236 143 72 228
OsCYP49 78 55 89 25 39 118 167 364 113 263
OsCYP50 444 90 96 108 49 103 54 156 123 50 124
OsCYP51 91 76 362 257 289 111 189
OsCYP57 220 179 99 104 145 223 40 208 141 141
OsCYP58 246 122 40 108 177 218 133 224 154 84
OsCYP63 98 107 108 98 51 92 100 120 69 47 56 41 106 133 421
OsCYP65 98 105 118 51 92 100 120 78 47 56 41 106 133 550
OsCYP70 81 55 89 25 39 163 155 328 113 145 309 81 360
OsCYP73 245 86 89 240 122 76 107 177 164 133 224 154 84
The sizes of exons were calculated based on gene structures generated with gene structure display server program (http://gsds.cbi.pku.edu.cn).
140
5.3.6 Cyp-encoding DNA sequences in wheat
A search of TIGR Plant Transcript Assemblies database (http://plantta.jcvi.org) using
the sequences of Arabidopsis Cyp loci (Romano et al., 2004a) as queries led to
identification of twenty-two Transcript Assemblies (TAs) ranging from 742 to 2237 bp
(Table 5.3). The TAs encoded 22 putative proteins, 15.5-70 kDa in size including any
predicted signal peptides. Their subcellular locations were predicted by two programs
(as for rice). Based on this information, the wheat Cyps could also be grouped into five
groups (Fig 5.2), i.e. cytosolic (10), ER (3), chloroplast Cyp (5), nuclear (1), and
mitochondrial (3). Their key characteristics are presented below. The putative amino
acid sequences and domains were compared to those in rice and other plants; this
information is presented later (section 5.3.7).
The ten putative full-length cytosolic proteins ranged from 148 aa/16 kDa (TaCYP16-2)
to 389 aa/46.2 kDa (TaCYP46-1). Two TAs (TA87300_4565, TA77265_4565)
encoding TaCYP31-2 and TaCYP46-1 respectively, had no stop codon, while
CV781674 encoding TaCYP21 had no start codon. TaCYP18-1 has been reported as
TaCYP18-3 on chromosome 6AS (Johnson and Bhave, 2004a). Eight members
(TaCYP16-2, TaCYP17, TaCYP18-1, TaCYP18-2, TaCYP19, TaCYP21, TaCYP42-2,
TaCYP31-1) contained only the main domain for PPIase activity (Galat, 1999). Two
members (TaCYP31-2, TaCYP46-1) were multi-domain. TaCYP46-1 contained the
TPR at C-terminus, required for association with the Hsp90 chaperone complex (Duina
et al., 1996) and TaCYP31-2 had the Arg/Lys rich domain at N-terminus, characteristic
of RNA-interacting proteins (Fu, 1995).
The three putative full-length ER-located proteins (including SPs) ranged from 213
aa/22.8 kDa (TaCYP23-1) to 231 aa/25.8 kDa (TaCYP26-2) (Table 5.3). TaCYP23-1
was 100% identical to TaCypB7A on coding sequence level and its gene was located on
7AL (Chapter 3). All members had ER-targeting signals at N-terminus (Fig 5.4) and a
SP, with cleavage site predicted to be between G34 and K35 (TaCYP23-1), A28 and
A29 (TaCYP24-1) and A24 and S25 (TaCYP26-2). The mature proteins were thus
estimated to be 19.2-23.4 kDa.
The five putative full-length chloroplast localized proteins ranged from 240 aa/25.7 kDa
(TaCYP26-1) to 423 aa/46.3 kDa (TaCYP46-2). TA106818_4565 had no start codon,
141
suggesting its product TaCYP32 is partial at N-terminus. The N-terminal bipartite
chloroplast/thylakoid targeting signal (C/T) for translocation into the thylakoid lumen
(He et al., 2004; Romano et al., 2004a), as predicted by the ‘TargetP 1.1 Server’, existed
in four members (Fig 5.4). Alignment of TaCYP32 with TaCYP36 and rice Cyps
OsCYP46 and OsCYP50 showed highest similarity (described later); therefore,
TaCYP32 was placed in this group although it lacked a C/T. TaCYP46-2 contained the
leucine-zipper motif between the C/T signals and main domain (Fulgosi et al., 1998).
The three putative full-length mitochondrial proteins ranged from 135 aa/ 15.5 kDa to
233 aa/26.2 kDa and contained N-terminal mitochondrial targeting signal (Fig 5.4).
One member was likely nuclear, the full-length protein being 636 aa/70.0 kDa. It
contained the Arg/Lys rich domains with SR rich motifs being considered as nucleus
localized signals (NLS), a characteristic of RNA-interacting proteins (Fu, 1995).
Table 5.3 Cyclophilin family in Triticum aestivum*
Namesa TA ID
TA (bp) ST-SP
b Exon RFWH
c
Protein (KDa)/aa
Cellular location
d
TaCYP16-1 CV777661 781 180-587 n/a HW-- 15.5/135 M
TaCYP16-2 TA87618_4565 810 75-521 n/a RFHY 16.0/148 Cytosol
TaCYP17 CK159156 694 104-547 n/a RFSH 16.5/147 Cytosol
TaCYP18-1 TA51537_4565 938 188-682 1 RFSH 18.2/164 Cytosol
TaCYP18-2 DR738025 931 130-645 n/a RFWH 18.4/171 Cytosol
TaCYP19 DR739063 1152 146-685 n/a RFWH 18.7/179 Cytosol
TaCYP21 CV781674 742 n/a-587 n/a RFHH 21.3/194 Cytosol
TaCYP23-1 CK208443 1061 67-708 7 RFWH 22.8/213 ER
TaCYP23-2 TA86901_4565 855 203-826 n/a HLDI 23.1/207 M
TaCYP24-1 TA85832_4565 1073 131-781 n/a RFWH 23.6/216 ER
TaCYP24-2 TA75912_4565 1267 194-841 n/a RFHH 23.9/215 Cytosol
TaCYP26-1 TA79896_4565 1062 104-826 n/a RFWH 25.7/240 CP
TaCYP26-2 TA85549_4565 897 46-741 n/a RFHY 25.8/231 ER
TaCYP26-3 DR738857 1008 96-833 n/a RFWH 25.9/245 CP
TaCYP26-4 TA86902_4565 1040 14-715 n/a HSDI 26.2/233 M
TaCYP31-1 TA77268_4565 947 106-n/a n/a RFHH 30.7/280 Cytosol
TaCYP31-2 TA87300_4565 1223 87-935 n/a RFWN 31.4/282 Cytosol
TaCYP32 TA89563_4565 1145 39-986 n/a SING 32.2/315 CP
TaCYP36 TA106818_4565 1176 n/a-992 n/a CING 35.7/329 CP
TaCYP46-1 TA77265_4565 1288 122-n/a n/a RFHH 46.2/389 Cytosol
TaCYP46-2 TA67068_4565 1655 127-1398 n/a RFNG 46.3/423 CP
TaCYP70 TA75909_4565 2237 145-2055 n/a RFHH 70.0/636 Nucleus
*As identified in TIGR Plant Transcript Assemblies database (http://plantta.jcvi.org, last accessed 02/2009).
aCyps named based on molecular weight of putative proteins; the names in
bold are reported by other names, e.g. TaCYP18-1 reported as TaCYP18-3 (Johnson and Bhave, 2004b); TaCYP23-1 as TaCypB-A;
bST-SP, translated region from start codon (ST) to
stop codon (SP); cFour residues essential for PPIase activity and CsA binding.
dcellular location
predicted by ‘PSORT Prediction’ (http://psort.ims.u-tokyo.ac.jp), ‘TargetP 1.1’
(http://www.cbs.dtu.dk/services/TargetP), M: mitochondria; CP: chloroplast.
142
70/135TaCYP16-1
148TaCYP16-2
147TaCYP17
164TaCYP18-1
171TaCYP18-2
179TaCYP19
194TaCYP21
24 213TaCYP23-1
70 207TaCYP23-2
34 216TaCYP24-1
1 179TaCYP24-2
48 240TaCYP26-1
51 245
29 198TaCYP26-2
TaCYP26-3
70 233TaCYP26-4
1 218TaCYP31-1
1 169TaCYP31-2
80 315TaCYP32
112 329 TaCYP36
TaCYP46-11 192
223 432TaCYP46-2
1 179TaCYP70
ER
C/T
MTS
C/T
MTS
C/T?
C/TER
ER
L/Z97 156
TPR TPR TPR
282 345
R/K
199/282
R/K
230 636
NLS
MTS
Fig 5.4 Structure of putative Cyps proteins in wheat. Grey box indicates main domain for PPIase catalysis; ER: ER-targeting signal; C/T: chloroplast/ thylakoid lumen targeting signal; MTS: mitochondrial targeting signal; TPR: tetratricopeptide repeat; R/K: Arg/Lys rich domain; dark bars in the R/K domain indicate NLS (nuclear localisation signal). L/Z: leu zipper.
5.3.7 Comparative analyses of putative Cyp amino acid sequences of rice and
wheat
The putative amino acid sequences of cytosolic single domain Cyps, translated from the
CDSs (exon contigs) in rice and TAs in wheat, were aligned with these types of Cyps in
Arabidopsis (Romano et al., 2004a; He et al., 2004) and CypA in human (hCyP-18a;
NP_066953) (Fig 5.5A). Three residues, corresponding to R55, F60 and H126 in
hCypA essential for PPIase activity (Zydowsky et al., 1992) (Table 5.4) were conserved
in seven out of nine Cyps in rice and seven out of eight Cyps in wheat. Residues (Q33-
I38-Y98; R44-F49-Y115; R32-F37-Y103, respectively) in OsCYP16, OsCYP18-1 and
TaCYP16-2 replaced these three residues. The W121 in hCypA essential for CsA
binding (Bossard et al., 1991) was conserved in five rice Cyps and two wheat Cyps. It
was replaced by H in OsCYP18-1, TaCYP16-2, TaCYP24-2, TaCYP31-1, TaCYP21;
or S in OsCYP18-2, OsCYP19-3, OsCYP20, TaCYP17, TaCYP18-1 (summaried in
Tables 5.1, 5.3).
143
Table 5.4 Reported functionally important residues in human CypA
Function Residues Reference
PPIase activity R55, I57, F60, Q63, A101, N102, Q111, F113, L122, H126, R144
Kallen and Walkinshaw, 1992
Essential for PPIase activity R55, F60, H126 Zydowsky et al., 1992
CsA contact R55, F60, M61, Q63, G72, A101, N102, A103, Q111, F113, W121, L122, H126
Pflügl et al., 1993
Essential for CsA binding W121 Bossard et al., 1991
Calcineurin (CN) binding R69, K125, R148 Rascher et al., 1998
Essential for CN binding R69 Rascher et al., 1998
N-linked glycosylation N71, N108, Hirtzlin et al., 1995; Pelle et al., 2002 O-linked glycosylation T73, S110
The seven amino acid insertion located between α-helix-II and β-strand-III (Saito et al.,
1999; Johnson and Bhave, 2004b) was absent in some of the Cyps, present in
OsCYP22-1, TaCYP31-1, TaCYP21 (4 aa); OsCYP18-3, OsCYP19-2, TaCYP18-2,
TaCYP19 (7 aa) and OsCYP19-1 and TaCYP24-2 (8 aa). The phylogenetic relationship
of these single domain Cyps was determined by analysis of the alignments with the
Neighbor-Joining program in MEGA 4.0 (http://www.megasoftware.net) (Fig 5.6A).
The possible orthologues TaCYP16-2 and OsCYP18-1 were >76% identical to
AtCYP18-1. TaCYP17 and TaCYP18-1 were grouped together and their amino acid
sequences were conserved in the comparable parts with only 17 aa extension at the N-
terminus in TaCYP18-1. They may be encoded by allele genes or by one gene with
different splice forms. OsCYP18-2, OsCYP19-3 and OsCYP20 were grouped together,
and were encoded by exons contigs produced by different splice forms of
LOC_Os08g44520. All five orthologues, TaCYP17, TaCYP18-1, OsCYP18-2,
OsCYP19-3 and OsCYP20, were >75% identical to AtCYP18-2. Likewise, TaCYP18-
2 and OsCYP81-3 were >75% identical to AtCYP18-3, AtCYP18-4, AtCYP19-1 and
AtCYP19-2; and TaCYP19 and OsCYP19-2 had >75% identity to AtCYP19-3.
TaCYP21 and TaCYP31-1 were grouped together and their amino acids sequences were
almost conserved in the comparable part with only four-residue variations. TaCYP31-1
contained extensions at both the N- and C-termini. TaCYP21 and TaCYP31-1 may be
encoded by allelic genes or the same gene with different splices forms.
The sequences of cytosolic multi-domain Cyps were also aligned (Fig 5.5B). Residues
corresponding to R55, F60 and H126 of hCypA were conserved in five Cyps in rice and
one Cyp in wheat; OsCYP57 and TaCYP31-2 had R-F-N instead. W121 in hCypA
was conserved in three of six Cyps in rice and one of two in wheat; OsCYP43,
144
OsCYP44, OsCYP51 and TaCYP46-1 contained H instead (Tables 5.1, 5.3). The
sequences exhibited a 6-8 aa insertion between α-helix-II and β-strand-III. At the N-
terminus, OsCYP73 contained WD40 of the same size as AtCYP71, while OsCYP58
contained a partial WD40, with 86 aa deletion. OsCYP43, OsCYP44 and TaCYP46-1
had an identical TPR (21 aa) to AtCYP40 (He et al., 2004). OsCYP57 and TaCYP31-2
contained an Arg/Lys domain analogous to that in Arabidopsis. The phylogenetic
relationships were assessed by analysis of the alignments (Fig 5.6B). TaCYP31-2 and
OsCYP57 had >60% identity to AtCYP57 in overlapping part and TaCYP46-1,
OsCYP43 and OsCYP44 had >64% identity to AtCYP40. OsCYP58 and OsCYP73 had
67% identity to AtCYP71 and OsCYP51 had 55% identity to AtCYP65. Compared to
AtCYP65, OsCYP51 was short of the U-box.
The putative amino acid sequences of ER-Cyps were aligned with those in Arabidopsis,
microsomal Cyp in maize (AY103896) and hCyP-18a (Fig 5.5C). R55, F60 and H126
in hCypA were conserved in four Cyps in rice and two in wheat; OsCYP26-1 and
TaCYP26-2 contained R-F-Y. W121 of hCypA was conserved in three rice and two
wheat Cyps; OsCYP22-2 contained R and OsCYP26-1 and TaCYP26-2 contained H
instead. Four out of five (OsCYP22-2, OsCYP23, OsCYP24-1, OsCYP24-2) Cyps in
rice and two of three (TaCYP23-1, TaCYP24-1) in wheat contained a 7 aa insertion
between α-helix-II and β-strand-III. All contained the N-terminal ER-targeting signals
of variable lengths and C-terminal ER retention signals with four (Freedman et al.,
2002), eleven (Boldbaatar et al., 2008), or thirty-six residues. The phylogenetic
relationships (Fig 5.6C) showed the possible orthologues TaCYP23-1, OsCYP24-1 and
OsCYP24-2 had >73% identity to AtCYP19-4 and AtCYP20-1; TaCYP24-1 and
OsCYP23 had >64% identity to AtCYP21-1, and TaCYP26-2 and OsCYP26-1 had
>69% identity to AtCYP23-1.
The putative amino acid sequences of chloroplast Cyps (Fig 5.5D) showed residues
corresponding to R55, F60, H126 and W121 of hCypA were conserved in only two
Cyps (OsCYP25, OsCYP27) in rice and two (TaCYP26-1 and TaCYP26-3) in wheat
(Tables 5.1, 5.3). These sequences had no insertion between α-helix-II and β-strand-III;
however, six Cyps (OsCYP28, OsCYP31, OsCYP40, OsCYP46, OsCYP47, OsCYP50)
in rice and three (TaCYP32, TaCYP36, TaCYP46-2) in wheat contained insertions of
11-24 residues. All contained N-terminal C/T targeting signals of variable lengths and
145
TaCYP46-2 and OsCYP47 contained Leu-Zipper similar to AtCYP38. The
phylogenetic relationships are shown in Fig 5.6D. TaCYP26-1 and OsCYP25 had
>54% identity to AtCYP20-3, and TaCYP26-3 and OsCYP27 had >59% identity to
AtCYP20-2. TaCYP32 and TaCYP36, with 71% identity, may be encoded by duplicate
genes. TaCYP32, TaCYP36, OsCYP46 and OsCYP50 had >62% identity to AtCYP37;
and TaCYP46-2 and OsCYP47 had 72% identity to AtCYP38.
The putative nuclear Cyps (Fig 5.5E) R55, F60, H126 of hCypA were conserved in
three of four Cyps in rice and in TaCYP70; OsCYP63 contained K-F-H. W121 was
substituted by H (OsCYP70, TaCYP70), Y (OsCYP63, OsCYP65) or R (OsCYP49)
(Tables 5.1, 5.3). OsCYP49, OsCYP70 and TaCYP70 contained an 8 aa insertion
between α-helix-II and β-strand-III. Alignment with AtCYP59 showed OsCYP63 and
OsCYP65 contained RRM and zinc-finger motifs involved in RNA-binding and
protein-protein interaction (He et al., 2004). All contained the Arg/Lys rich domains of
variable sizes at C-terminus. TaCYP70 and OsCYP70 had >69% to AtCYP63 in the
main domain part corresponding to hCypA (Fig 5.5E). The phylogenetic relationships
(Fig 5.6E) show the orthologues of TaCYP70 may be OsCYP70 and AtCYP63. The
orthologue of OsCYP63 and OsCYP65 (encoded by same locus; section 5.3.1) may be
AtCYP59, with an identity of >58% in the comparable parts including main domain,
RRM motif, and zinc-finger motif.
The mitochondrial Cyps (Fig 5.5F) showed variable residues in place of to R55, F60,
H126 (OsCYP26-2: H-F-L; TaCYP23-2: H-L-I; TaCYP26-4: H-S-I) (Tables 5.1, 5.3).
TaCYP16-1 contained a partial main domain corresponding to the first 68 aa of hCypA.
All mitochondrial Cyps in rice and wheat contained a D corresponding to W121 and a 4
aa deletion between α-helix-II and β-strand-III. The mitochondrial targeting sequences
at N-terminus had conserved lengths. The phylogenetic relationship (Fig 5.6F) showed
all three wheat Cyps grouped together. Compared to TaCYP26-4, TaCYP16-1 and
TaCYP23-2 had deletions of 98 aa and 26 aa respectively at the C-terminus, with a few
variable residues in comparable parts. Duplicated genes may encode three
mitochondrial Cyps in wheat.
146
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
hCypA ---------------------------------MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGE-KGFG--------YKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYG------------EKFEDEN-FILKHT-G
AtCYP18-1 ------------------------------------MS.TLHTNLG-----DIKC.I.C.E...S....L..C-----AS.-------Y.D.TI...N.K...I....PK-GT.K..T..W.------------K..N..IRDS...N-A
AtCYP18-2 ---------------------------MSARPEGSP.E.TLETSMG-----PFTV.MYYKHS.R.CR..LE..-----RR.-------Y.DNVL....VKD.IV....P.-GT.R..E....------------S.....INKE....-.
AtCYP18-3 ---------------------------------.AF.K.Y..MTI..Q.A..IVM..YT..T.R........C...-..V.GT-GKPLHF...K...V..N.........AG.....E....------------S......-.ER...-.
AtCYP18-4 ---------------------------------.S..R....MSLS.T.I..IEM.....TT.N........C...-..M.KL-GKPLHF...I...V............AK.....E....------------A..K...-..K...-.
AtCYP19-1 --------------------------------MAT..K.Y..MT.G.KSA..IVM..Y..TT.E........C...-R.I.KQ-GKPLH....S...V..K.........AG.....E....------------S..K...-..K...-.
AtCYP19-2 --------------------------------MASH.K....MTIG.A.A.KIVM..YT..T..........C...-..V.RS-GKPLHF...S...V..N.........KG.....E....------------A......-.ER...-.
AtCYP19-3 ---------------------------------.A..K.....LIGKMKA...VM.....VT.R..N.....C...-N.I.KA-GKALH....A.................G.....E....------------S......-.K....-.
AtCYP22-1 MNSGGGIVAAAAPS-----SGGGNVEWHVRPPNPK..V....VSIG.I.A..IKM.....IA........QFC...LRKA.KP----LG..ECQ...V.KD..V.S...LKND.S.CM....------------H......-.TA...-.
AtCYP26-1 ---------------------------------.A..K....LT...K.A..IVI.....LT.R......G.C...-R.I.KC-GKPIH....T.DH.V.DL.WC...IIFE.----EP.HS------------.ELD..Y-...N.ED.
OsCYP16 ------------------------------------------------------M..Y..L..L........Y.KK----------PLH...L.LSQHY.RIHVE.RRLRQGPWHRRRVHLR------------RHLPN..-.L.P.D--
OsCYP18-1 ------------------------------------MS.TLHTNLG-----DIKC.V.C.QA.R.....L..C-----AS.-------Y.D.TI...N.K...I....P.-GT.K..T..W.------------K..A..FRES...N-A
OsCYP18-2 ---------------------------MWGSADGGT.E.TLETSMG-----AFTI.MYYKHA...CR..LE..-----RR.-------Y.DNVI.....KD.IV....P.-GT.R..E....------------A.....IRPE....-.
OsCYP18-3 ---------------------------------.S.TR....MT.G.A.A..IVM..Y.KD..R........C...-..V.KS-GKPLH....T...V..E..........G.....E....------------...A..V-.KF..D-S
OsCYP19-1 --------------------------MAPAASSKS..R..L...IG..WV...VI..L.....D......R.C..--ERA.RSGKSRLH....A...VV.........I.AG.....E.ALDG---------AARH.P..G-.AV..D-.
OsCYP19-2 -------------------------------MASK..K.....LIGKARA...VM.....T.........C.C...-..L.AS-GKPLH....A......N..........G.....E....------------DR.A...-.K.R..-.
OsCYP19-3 ---------------------------MWGSADGGT.E.TLETSMG-----AFTI.MYYKHA...CR..LE..-----RR.-------Y.DNVI.....KD.IV....P.-GT.R..E....SAYFLLSSSFNLA.....IRPE....-.
OsCYP20 ------------MDPRSKADRIVRRTAMIGAATAAYFLLTA.YGP------DYPNPMYYKHA...CR..LE..-----RR.-------Y.DNVI.....KD.IV....P.-GT.R..E....------------A.....IRPE....-.
OsCYP22-1 MASSGGAAISAGPTPPSAAAASSSVDWHLRPPNPK..V....VTIGSI.A..IKM.....I.........QFC...HRKS.LP----QG...CQ...V.KD..I....YMKGD...CT....------------T..D...-..A...-.
TaCYP16-2 -----------------------------------------------------MV.V.CEQ..R.....L..C-----AS.-------Y.D.TV...N.K...V....P.-GT.K..A..W.------------G..A..FREA...G-A
TaCYP18-1 ---------------------------MWGGDDGGT.E.TLETSMG-----SFTV.MYHKHA...CR..VE.A-----RRK-------Y.DNVV.....KD.IV....PS-GT.R..E....------------A.....IRPE....-.
TaCYP17 --------------------------------------------M.-----PFTV.MYHKHA...CR..VE.A-----RRK-------Y.XNVV.....KD.IV....PS-GT.R..E....------------A.....IRPE....-.
TaCYP18-2 ---------------------------------.A..R....MT.G.A.A..IVM..YK.A..R.V......C...-..V.KS-GKPLH....A...V..D..........G.....E....------------...A..K-.VH...-K
TaCYP19 ------------------------------MAAAK..K.....LIGQAKA...VM..Y.....R..A...Q.C...-..L.AS-GKPLH....A.................G.....E.V..------------A..A...-.L.R..-.
TaCYP24-2 ---------------------------MPKFKKNL..Q..LE.SI..R.AE.IT......V...........C...-..L.ESTKKPLYF..THI........A.A...S.GD.R..E....------------G..P...-.K...D-Q
TaCYP31-1 ---MEGGGEGAAPAPAAAAAEVKNPRCFMDITIGGEMEGRIV.ELY-----ASVVPRT.ENFRAHCTGEKGVG-----AS.KP----LH....Y.....K...V......AGD....E....------------S....K.-.....E-R
TaCYP21 --------------------------------------------------------------TRPCTGEKGVG-----AS.KP----LH....Y.....K...V......AGD....E....------------S......-.....E-R
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
βI βII αI αII βIII βIV
+ + + + # -*-* * *
++ + + + +
A 7 aa
147
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
hCypA PGILSMANAGPNTNGSQFFICTAKT-EWLDGKHVVFGKVK-EGMNIVEAMER-FGSR-NGKTSKKITIADCGQLE---------------------------------------------------------------------------
AtCYP18-1 R.M.....S...........TY..Q-PH.N.LYTI....I-H.FEVLDI..K-TQTGPGDRPLAE.RLNRVTIHANPLAG----------------------------------------------------------------------
AtCYP18-2 A...................TL.PQ-PS.....TI..R.C-R..EVIKRLGS-VQTDNTDRPIHEVK.LRTKVID---------------------------------------------------------------------------
AtCYP18-3 ..........A...........V..-D..........Q.V-..LDV.K.I.K-V..S-S..PT.PVVV......S---------------------------------------------------------------------------
AtCYP18-4 A.......S.............D..-S..........Q.V-K.LDV.K.I.K-V..D-S.....VV..T.....S---------------------------------------------------------------------------
AtCYP19-1 ..........A...........E..-S..........Q.V-..L.V.RDI.K-V..D-S.R...PVV......IS---------------------------------------------------------------------------
AtCYP19-2 ..........A...........V..-D..........Q.V-..LDV.K.I.K-I..S-S..PT.PVV.....EISS--------------------------------------------------------------------------
AtCYP19-3 ........S.............E..-S............V-D.Y.V.K...D-V..D-M.NP.ERVV.E...E.KNPSS-----------------------------------------------------------------------
AtCYP22-1 ..L.....S......C....TC..C-D...N......R.LGD.LLVMRKI.N-VAIGP.NRPKLAVV.TE..EM----------------------------------------------------------------------------
AtCYP26-1 ...I...DS----.....Q.HMKDYGLQV..D...I...V-..LDLMRNI.KEVITTTTRTP..PVV.....E.SDYRSERCYLMKNIEKEVIIKTAKDNKPVVIADCGGLSDDRSERYYLINIVVACMVLMCFWSWFV-----------
OsCYP16 --RPWLP.GSAEN.I....TR----VP.F..NY....CII-S.FHNLK.I.AEVEVKIANRGEVV.VPPPSLTTN---------------------------------------------------------------------------
OsCYP18-1 R.VM....S...........TY..Q-PH.N.HYT..A..I-H.FEVLDL..K-AQTGPGDRPLAE.RLNRVTIHANPLAN----------------------------------------------------------------------
OsCYP18-2 A...................TL.PC-QS.....TI..R.S-K..E..KRLGS-VQTDKSDRPIHEVK.LRTVVKD---------------------------------------------------------------------------
OsCYP18-3 ......................VPC-S..........R.V-...DV.K.I.K-V...-G.S.A.PVV.......S---------------------------------------------------------------------------
OsCYP19-1 ..VV................TVD.A-P....R..A..R.V-A..GA.R.ID.-T.TW-S...V.PVV.T...V.----------------------------------------------------------------------------
OsCYP19-2 ..V...................TR.-T............V-D.YTV..K..Q-V..G-S.G.AERVL.E.....ADDHAN----------------------------------------------------------------------
OsCYP19-3 A...................TL.PC-QS.....TI..R.S-K..E..KRLGS-VQTDKSDRPIHEVK.LRTVVKD---------------------------------------------------------------------------
OsCYP20 A...................TL.PC-QS.....TI..R.S-K..E..KRLGS-VQTDKSDRPIHEVK.LRTVVKD---------------------------------------------------------------------------
OsCYP22-1 ..L.....S.V.S.......TC..C-....N......R.LGD..LA.RKI.N-VATGP.NRPKLACV.SE..EM----------------------------------------------------------------------------
TaCYP16-2 R.T.....S...........TY..Q-PH.N.HYT..A..I-H.FDVLDL..K-TPTGPADRPLAE.RLNRVTVHANPLAG----------------------------------------------------------------------
TaCYP18-1 A...................TL.PC-QS.....TI..R.C-R..E..KRLGS-IQTDK.DRPIHEVK.LRTVVKD---------------------------------------------------------------------------
TaCYP17 A...................TL.PC-QS.....TI..R.C-R..E..KRLGS-IQTDK.DRPIHEVK.LRTVVKD---------------------------------------------------------------------------
TaCYP18-2 ......................VPC-N..........E.V-...DV.KNI.K-V...-S.TC..QVV.......----------------------------------------------------------------------------
TaCYP19 ..V........G.............-P..........Q.V-D.YGV..K..A-V..S-G.S.AQPVV.E.....PDDQG-----------------------------------------------------------------------
TaCYP24-2 ........S.E.........TFKAV-PH...........L-N.KALLKKL.A-L..E-S..PTCPVK.V...EASNIDTQNQLHGEKEKKLKRAVEDNQRCWREGQNQKNI---------------------------------------
TaCYP31-1 K.......S...........T.TR.-PH........AR.I-K..GV.RSC.H-IPVGEADRPTVDAV..E..E.PEGADDGVVNFFKDGDMYPDWPNDLDEKPTEVSWWMEAVESAKAFGNDNFKKQDYKTALRKYRKALRYLDVCWETE
TaCYP21 K.......S...........T.TR.-PH........AR.I-K..GV.RSC.H-IPVGEADRPTVDAV..E..E.PEGADDGVVNFFKDGDMYPDWPNDLDEKATEVSWWMEAVEAAKAFGKDNFRN-------RIIRRP-----------
βV βVI βVII αIII βVIII
++ * * *
+ + - - * *
+ + * * # * + #
148
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP43 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP44 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCYP46-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP57 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP57 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCYP31-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP71 -------------------MEEESKNGGTTIPTEELAVVAVPPVVEEEEPMVGPGPAP-RGKRKRPLQFEQAYLDSLPSANMYEKSYMHRDVVTHVAVSAAEFFISGSMDGHLKFWKKKGVGIEFAKHFRSHLGPIEGLAVSIDGLLCCT
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------MQVSVDGLLCCT
OsCYP73 MATASDAPASSTITTATDDAEVERDQGNGNGAVSAAPAAVGKEAAAEEEEMIGPAPVPPRPRKKRPLQFEQAFLDALPSAAMYEKSYMHRDVVTHVAVSPADYFITGSADGHLKFWKKKPAGIEFAKHFRSHLSPIEGLAVSVDGLLCCT
AtCYP65 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP51 ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP43 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP44 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCYP46-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP57 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP57 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCYP31-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP71 ISNDHAVKIYDVVNYDMMAMIRLPYIPGAVEWVYKQGDVKAKLAVSDRDSLFVHIYDPRSGSNEPIASKEIHMNPIKVMKYNPVSDTMISGDTKGIIEYWSATTLQFPEDEVNFKLKSDTNLFEIIKCKTTISAIEVSPDGKQFSITAPD
OsCYP58 ISSDWSVKIYDVVNYDMMFMMRLPFVPGAIEWVYRQGDVKPKLAVSDRNTPFVHIYDTHSGSNDPIISKEIHAGPVKVMKYNHVHDVVISADAKGLLEYWSPSTLKFPEDAVNFRLKTDTNLFEIAKCKTSVSAIEMSNDGTQFVVTSPD
OsCYP73 ISSDWSVKIYDVVNYDMMFMMRLPFVPGAIEWVYRQGDVKPKLAVSDRNTPFVHIYDTHSGSNDPIISKEIHAGPVKVMKYNHVHDVVISADAKGLLEYWSPSTLKFPEDAVNFRLKTDTNLFEIAKCKTSVSAIEMSNDGTQFVVTSPD
AtCYP65 MGKKQHSKDRMFITKTEWATEWGGAKSKENRTPFKSLPYYCCALTFLPFEDPVCTIDGSVFEITTIVPYIRKFGKHPVTGAPLKGEDLIPLIFHKNSEGEYHCPVLNKVFTEFTHIVAVKTTGNVFCYEAIKELNIKTKNWKELLTEEPF
OsCYP51 ----------------------------------------------------------------------------------------------------------------------------MRWGLAIQELNIKPKNWRELLTDEPF
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP43 ---------------------------------------------------------------------------------------------------------------------------------------------------MEG
OsCYP44 ---------------------------------------------------------------------------------------------------------------------------------------------------MEG
TaCYP46-1 ---------------------------------------------------------------------------------------------------------------------------------------------------MEG
AtCYP57 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP57 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCYP31-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP71 RRIRVFWFRTGKLRRVYDESLVVAQDLQRSDAPLYRLEAIDFGRRMAVEKELEKTESAPQPNAVFDESSNFLIYATFLGIKVINLHTNTVARILGKVESNERYLRVALYQGDQGGKKVRKIPAAAANVNESKEPLTDPTILCCAFKKHRI
OsCYP58 RRIRVFWFKTGKLRRVYDESLEVAQDLQKSDIPMYRLDAIDFGRRMAVEKEIEKTENVPQPNAVFDESSNFLIYATLLGIKIINLHTNKVSRILGKVENNERFLRIALYQGDKGNKKVRKIPSVAANVNDSKEPLSDPTLLCCAFKKHRI
OsCYP73 RRIRVFWFKTGKLRRVYDESLEVAQDLQKSDIPMYRLDAIDFGRRMAVEKEIEKTENVPQPNAVFDESSNFLIYATLLGIKIINLHTNKVSRILGKVENNERFLRIALYQGDKGNKKVRKIPSVAANVNDSKEPLSDPTLLCCAFKKHRI
AtCYP65 TRADLITIQNPNAVDGKVTVEFDHVKNGLKIDDEELKKMNSDPAYNINVSGDIKHMLADLGTDKAKEIALHGGGGNKARNERAAAIAAILESRSKIKEVS-KAEQPKQTYSVVDAASASVFGRSADAAKAGSSDKTAARIAMHMAGDRTP
OsCYP51 TRNDLITIQNPNAVDSKILGEFDHVKKGLKLEDEELQRMKNDPTYNINISGDLKQMIKELGTEKGKLAFLHGGGGQKAQKERAAALAAILAKKEKDDSKSGKEPKPHQPFSIVDAASASVHGRSAAAAKAATAEKTAARIAMHMAGDRAP
B
WD40
WD40
149
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA --------------------------MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKG--------FGYKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGP--GILSMANA-G-PNTNGSQF
AtCYP40 --------------------------.GRSKC.M..SIG..LE..IVI..YD.V.........L.C.....LGPNTGVPLH...NR...V.K...I....ISAND....E....L..D....E...ERK--.M.....S-.-........
OsCYP43 GGEGAESAAVASAAAA-------EVEVK..RC.M.VSIG..IE..IVI..Y.SV..R........C.....VGAVTGKHLH........V.K...V....I.AGD....E....L.......V...ERK--.M.....S-.-........
OsCYP44 GGEGTTTAAAAAAVAAGKEEEAEVVVVR..RCYL.VSIG.DME..IVV..Y.SVA.R........C.....VSAATGVPLH.....I...VK...V....I.AGD....E....LN......V...ERK--.M......-.-.D......
TaCYP46-1 GGEGAAPAPAAATAAE----------VK..RC.M..TIG..ME..IVI..Y.SV..R........C.....VGAS-GKPLH....Y.....K...V......AGD....E....S...........ERK--.......S-.-........
AtCYP57 -------------------------------MSTVYVLEPPTK.K.IVNTTHGPIDVELWPKE.PKSVRNFVQLCLE--GYFDNTI...V....LV....P.-GS....D....GV.A..-.HSRLRFSHR..VA....SS-..S.....
OsCYP57 -------------------------------MSSVYVLEPPTK.K.VVQTT.GPLDIELWPKE.PKAVRNFVQLCLE--GY.D.TL...V.KS.LV....P.-GS....E....AP.A..-.HTRLRFNHR.LVAC...-.T.HS.....
TaCYP31-2 -------------------------------MSSVYVLEPPTK.N.VVQTT.GPIDIELWAKE.PKATRNFVQLCLE--GY.D.TL...V.KS.LI....P.-GS....E....AP.A..-.HSRLRFNHR.L.AC...-.T.HS.....
AtCYP71 YMFSRREPEEPEDASQGRDVFNEKPAADELMSVS..GNSATTSLPENVIMHTTLGDIHMKLYPEECPKTVENFTTHCRNGY.DNHL...V.R...I.T..PL-GD....Q..W.RE....-.HKSLRHDRPFT......-.-........
OsCYP58 YLFSRREPEEPEDATKGRDVFNEKPPPEELLAVS.LGKTATTSLPDNLVMHTSMGDIHLRLYPEECPKTVENFTTHCRNGY.DNLI...V.K...I.T..PL-GD....Q..W.RE....-.HKSLRHDRPFT......-.-........
OsCYP73 YLFSRREPEEPEDATKGRDVFNEKPPPEELLAVS.LGKTATTSLPDNLVMHTSMGDIHLRLYPEECPKTVENFTTHCRNGY.DNLI...V.K...I.T..PL-GD....Q..W.RE....-.HKSLRHDRPFT......-.-........
AtCYP65 VNSKMVKSRYSSGAASRSFTSSAFTPVTKNDFELIKVEKNPKKKGYVQFQTTH-GDLNI.LHCDIAPRACENFITLCERGY.N.VA...S.RN..I....P.-GT.K..E..W.KP.K..-PNS.LLHSGR.VV....S-.-.H......
OsCYP51 VNAKLVKSRYTTGAASRSFTSTAYDPVTKNELEYVKVEKNPKKKGYVQLHTTH-GDLNL.LHCDITPRTCENFLTHCENGY.N.LI...S.KN..I....P.-GT.S..E..W.KP.K..-LNS.LIHSGR.VV....S-.-.H......
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA FICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE-------------------------------------------------------------------------------------------------
AtCYP40 ..T.TR.SH.........R.TK..GV.RSI.HVSIEEQSCPSQDVVIHDCGEIPEGADDGICDFFKDGDVYPDWPIDLNESPAELSWWMETVDFVKAHGNEHFKKQDYKMALRKYRKALRYLDICWEKEGIDEETSTALRKTKSQIFTNS
OsCYP43 ..T.TR.PH.........R.IK..GV.RSV.HAPVGEADRPTSDVEIVDCGELPEGADDGVVNFFNDGDTYPDWPNDLDEKPMEVSWWMDAVESAKAFGNNNFKKQDYKAALRKYRKALRYLDVCWEKEDIDEEKSSALRKTKSIILTNS
OsCYP44 ..T.TR.PH.........R.IK..GV.RS..HVSVGESDRPITDIVIVDCGELPEGASDGVVNFFSDGDMYPDWPNDLEEKPAEISWWMTAVDSAKSFGNEYFKKKDYKTALKKYRKAMRYLDLCWEKEEIDEEKSSALRKTKSIILTNS
TaCYP46-1 ..T.TR.PH........AR.IK..GV.RSC.HIPVGEADRPTVDAVIAECGELPEGADDGVVNFFKDGDMYPDWPNDLDEKPTEVSWWMEAVESAKAFGNDNFKKQDYKTALRKYRKALRYLDVCWEKEEIDEEKSSALRKTKSIILTNS
AtCYP57 .FTLD.CD...K..TI....TGDSIYNLLRLGEVDTSKDDRPLDPAPKILSV.VLWNPFEDIVPRVLAKTSEESAAEIK-EPPTKPVKKLNLLSFGEEAEEEEKELA-VVKQKIKSSHDVLNDPRLLKAEASDKERNASESKEVLSVREA
OsCYP57 ..SLDRCD...K.NTI....TGDSIFNLLALADIETDKDDRPVYPQ-KILSV.VLWNPFDDIVPRQLKKTEPTAKGDIEGKSKKKAVKQLNVLSFGDEVEEEENEAASSVKDKIKSIHDVLDDPRFLKGEAPDEQLTKEQEDKKKETVQS
TaCYP31-2 ..TLDRCD...K.NTI....TGDSIFNLLALADIETDKDDRPVYPQ-KILSV.VLWNPFDDIVPRQLKKIQPAPKADAERKPEKKAVKQLNVLSFGDEVEEEENEAASFVKDKIKSIHDVLDDPRFLKVEPQVEQLSKKKEGEAGKKLIS
AtCYP71 ..T.VA.P...N..T...R.VK..DV.QGI.KVKTDKNDRPYQDVKILNVTVPKS-----------------------------------------------------------------------------------------------
OsCYP58 ..T.VA.P...N..T...R.VK..DV.QQI.KVKTDKNDKPYQDVKILNVTVPKT-----------------------------------------------------------------------------------------------
OsCYP73 ..T.VA.P...N..T...R.VK..DV.QQI.KVKTDKNDKPYQDVKILNVTVPKT-----------------------------------------------------------------------------------------------
AtCYP65 .VLYKSATH.NY..T...G.VG.LATLA...NVPVDESDRPLEEIKIIEASVFVNPYTELDEEEEKEKAEKEKNEDKDIEKIGSWYSNPGSGTTEAGAGGGGVGKYLKAMSSTATKDTKGSLDSDISTIGVSKKRKTTASASTGFKDFSS
OsCYP51 ..LYKSAPH.NF..T...M.VG.LTTLS...KVPVDDDDRPLEEIKILKVSVFVNPYTEPDEEEEEKAKEEEKKKDEDYDKVGSWYSNPGTGVAGSTSSGGGVGKYLKAR-------TAG--FADVVADDSNKKRKASVSN-VEFKDFSG
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP40 AACKLKFGDAKGALLDTEFAMRDEDNNVKALFRQGQAYMALNNVDAAAESLEKALQFEPNDAGIKKEYAAVMKKIAFRDNEEKKQYRKMFV-----------------------------------------------------------
OsCYP43 SACKLKLGDLKGALLDADFALRESEGNAKAFFRQGQAHIALNDIDAAVESFKHALELEPSDGGIKRELAAAKKKIADRRNQERKAFARMFQPSGKSDKDNEESK----------------------------------------------
OsCYP44 SACKLKLGDLKGALLDADFALREGEGNPKAFFRQGQARIALNDIDAAVESFKHALQLEPNDGGIKRELAAAKKKIADRRDQERKAFSRMFQPSGGSEKIDEENN----------------------------------------------
TaCYP46-1 SACKMKLGDLKGALLDADFALRETEGNAKAFFRQGQAHIALNDIDAAVESFQHALDLEPNDGTIKRELAAAKKKISNGKIWSARRTPGMFPTLR---KIPRENK----------------------------------------------
AtCYP57 LNAKKEAAQKDKSFSVSDTVGNSDDDDDGEDETKFDAKMRNQVLSRRKEIGDTPSKPTQKKKSSSLKGREESTQRSDAVSSEDEKPRMEKLSLKKKGIGSEAKAEHMEKGDTDLQLYNASERARQLHKLKKRRLQGNEDSVLAKLEKFKQ
OsCYP57 VREALVSKKSDFRELEHDSETDDYPDDENEEDFDNRMRSQILRKRRELGDIRSSETSKKTDKAHRKDKELPVHRSDDDNDDDNEDHQLTKSRKFSMKKKGIGSEASAERMSKGDANLQLLNPAEQEKHLQKQKRRRLQGREDETLAKLQK
TaCYP31-2 LSSWHSLRTVQDGRRSCVP-----------------------------------------------------------------------------------------------------------------------------------
AtCYP71 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP65 W-----------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP51 W-----------------------------------------------------------------------------------------------------------------------------------------------------
6-8 aa+ + + + * * * * # -* -
++ * * *
+ - - *
+ *
+ **
+ #*
+#
TPR
TPR
Arg/Lys rich domain
Arg/Lys rich domain
βI βII αI αIIαII βIII βIV βV
βVI βVII αIII βVIII
150
910 920 930 940 950 960 970 980 990
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .
hCypA --------------------------------------------------------------------------------------------
AtCYP40 --------------------------------------------------------------------------------------------
OsCYP43 --------------------------------------------------------------------------------------------
OsCYP44 --------------------------------------------------------------------------------------------
TaCYP46-1 --------------------------------------------------------------------------------------------
AtCYP57 SISAKPFTSSNEPVVLTSSSEPVDNKEEDLSDWKNVKLKFAPERGKDKMSRRDDPDAYMVVDPLLEKGKEKFNRMQAKQKRREREWSGKSLA
OsCYP57 FKASFLSKNPATGNTEKKTDEEDYTGWHSNRLTFEPDSSKDGMTRKDDPDDYVVVDPLLEKGKQKFNKMQAKLKRREREWAGRSLT------
TaCYP31-2 --------------------------------------------------------------------------------------------
AtCYP71 --------------------------------------------------------------------------------------------
OsCYP58 --------------------------------------------------------------------------------------------
OsCYP73 --------------------------------------------------------------------------------------------
AtCYP65 --------------------------------------------------------------------------------------------
OsCYP51 --------------------------------------------------------------------------------------------
Arg/Lys rich domain
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA -------------------------------------------------------------------------MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFG-------YKGSCFHRIIPGFMCQGGDFTRH
AtCYP19-4 -------MAKA----------------SFILLGTLFLFGAIASIQAK----------------------EDLKE.THK.Y..VEI..KSA...VIG..GKA...........C.....V.KSGKPLH....K......S..I......HG
AtCYP20-1 -------MASS----------------VTLLLWSLLLLGTLSAIQAKK-------------------SKENLKEITHK.Y..VEI..KAA..IVMG..GKT....V......C.....I.KNGKALH....S......S..L......HG
AtCYP21-1 MRREISFLLQPRCLL------------LLVALTIFLVFALFNTGKDEEK------------------QVIEDHEITNR..L.VDI..QR...IVIG.YGTV....V......C.....KTSSGKPLH...TP.....S..VI....IIHG
AtCYP21-2 -------MAITATR-----------LVSLTLLWIVVLFVTLALIQIKLTDVADPSVNEKILDAKLNQVGEDLEG.THK.Y...QIN.S.A..ILIG..GNI.........S.C.....V.NMGKPLYF...S.........I.......G
AtCYP23-1 -----MGITRNLILG-----------------LACLAFVSIAKA-----------------------LPHEPELGSAR.V.QTSY-----.DIE.GFYPTVA...VDHIFK.VRLG----------G.NTNH.F.VDK..VA.VA.VASG
OsCYP22-2 -------MAARETSR-----------HASLCLWLALVAATLSLAQAVES-------------------EAELTK.TTK.....TIN.K.A..IVMG..GNT..........IC.....L.KSGKPLS...TP.........I....TVSG
OsCYP23 -------MLRKVAVA------------FLACAALYLAFAAYS--RRESL------------------GEVRLPA.TNR.YL.VEI..QHI..IVIG.YG.V....VA.....C...E.I.HKGKSLH....R.........I....IV.G
OsCYP24-1 -------MAGSGWRRIPAVRRPPMSRPASVCLWIVLVAATLALAQ-------------------AKKSKADLTE.THK.Y..VEI..K.A...VMG..GKT...........C.....T.KSGKALHF...A......S..I......LG
OsCYP24-2 -------MAGSGWRRIPAVRRPPMSRPASVCLWIVLVAATLALAQVLIH--------------LAKKSKADLTE.THK.Y..VEI..K.A...VMG..GKT...........C.....T.KSGKALHF...A......S..I......LG
OsCYP26-1 ----MAALLRHAAAAA------------AAVVLLIAAAASTDGATSF--------------------YASDPNLGSAR.V.QTTH-----.DIE.GF.PHVA...V.HIYK.VRLG----------C.NTNH.F.VDK..VA.VASVVGG
TaCYP23-1 -------MAMRAWRRSAAARAP-----AHLCLWLALVAATLVLAQ---------------------GKKSNLSE.THK.Y...EI..K.A...VMG..GKA...........C.....M.NSGKPLH....S......S..I......LG
TaCYP24-1 -------MLRKAAVG------------FLACLVLYLAFSSYP--RSQRA------------------AYVQLPA.THR.YL.VEI..QNI..IVIG.YGEV....V......C.....D.PKGKPLH...TP.........I....IV.G
TaCYP26-2 ----MAGLLRHIAAA----------------LLFLLTAASTAGASTY--------------------YASDPNLGSAR.V.QLNH-----.DIE.GF.PHVA...V.HIFK.VQLG----------C.NTNH.F.VDK..VA.VA.VMGG
Maize -------METSGWRRRAAARAS--WSPATVYLWLALAAAALTLAQ----------------------AKKDLTE.THK.Y..VEI..K.A..IVMG..GKT...........C.....I.KSGKALH....T......S..L......LG
160 170 180 190 200 210 220 230 240 250 260 270 280
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .
hCypA N----GTGGKSIYGEKFEDENFILKHTGPGILSMANA-GPNTNGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMER-FGSRNGK---TSKKITIADCGQLE---------------------------------
AtCYP19-4 .----.M..E....Q..A....K.......V.....S-.ED........T.VT.S....R.......VQ..DV.YKI.A-E.KQS.T---PKS.VV...S.E.PL--------------------------------
AtCYP20-1 .----.M..E.......A....K.......F......-.QD........T.VT.S....R.......VT..DV.YKV.A-E.NQS.T---PKS.VV.V.S.E.PL--------------------------------
AtCYP21-1 D----.KSSD....GT.P....KIQ.SHA.MVA...T-..DS.......T.V.AS..E.E...L...IQ..DN.F.I.GGA.TYS..---PR..VV...S.EIPKDKWDEER-------------------------
AtCYP21-2 D----.R..E....D..A....K.......F.....S-..DS.......T.VT.S....H.......LS..EV.RKI.A-Q.QDS.V---PKANVI.FAS.EVSL--------------------------------
AtCYP23-1 RSAPMNEEQRKEAEK.IVG.FSDV..VR-.T...GRYDD..SAQ.S.SMLLGNAPH..RQYA.....TK.DETLSKL.EVPTR.E.IFVMPTER...LSTYYYDTKMESCEEERSVLKRRLQASFVEVERQRMKCFP
OsCYP22-2 .----...CD....GM.P....KIN.SA..L.....Y-AKD........T.V.LTR...........LS..DV.YKI.A-E..QS.T---PRS.VL.S.S.E.K---------------------------------
OsCYP23 D----.K.SE....GT.P.....V...H..VIA...S-..DS.....Y.T.I..S....E.....R.IQ..DY.Y.I.GGA.TY...---PR..VV.T.S.EIPKEKWAEEV-------------------------
OsCYP24-1 D----.R..E....T..A....KI......L......-.RD........T.VT.S............LS..DV.YKI.A-E.QQS.S---PKS.VV...S.E.PM--------------------------------
OsCYP24-2 D----.R..E....T..A....KI......L......-.RD........T.VT.S............LS..DV.YKI.A-E.QQS.S---PKS.VV...S.E.PM--------------------------------
OsCYP26-1 RTAPMNDEQQEEAEKSVVG.FSTV..VR-.....GRHSD..SG..S.S.LLGDAPH...QYA...RLTK.DDTLRKL.QLPTR.E.IFVMPIER.S.LSTYYYDVDLESCEAEKSILRRRLSESASEVERWRRKCFA
TaCYP23-1 D----.R..E....T..A....K.......Y......-.RD........T.VT.S............LS..DV.YKV.A-E.KQ..T---PKSIVV...S.EVPL--------------------------------
TaCYP24-1 D----.KARE....GT.P....SV...H..VVA...S-.TDS.....Y.T.I..G....E.....R.IQ..DY.Y.I.GGA.TY...---PR..VL.T.S.EIPKEKWGEEAD------------------------
TaCYP26-2 RKAPMNKEQEQQAEKSIVG.FSTV..VR-.....GRYSD.DSAS.S.S.LLGDAPH...QYA...R.TK.DDTLRKL..LPTHKE.IFVMPIER.E.LSTYYYDIDVESCEAEKSILKRRLSESASEVERWRRKCFA
Maize D----.R..E....M..A....KI......L......-.RD........T.VT.S............LS..DV.YKV.A-E.RQS.Q---PKS.VV...S.E.PM--------------------------------
ER-targeting sequence
7 aa+ + + + * * * *
C#
- * - ++ * * *
- - + + * *
+ + * * #*
+ #
ER-localization signal
βI βII αI αII βIII βIV
βV βVI βVII αIII βVIII
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA -------------------------------------------------------------------------MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFG-------YKGSCFHRIIPGFMCQGGDFTRH
AtCYP19-4 -------MAKA----------------SFILLGTLFLFGAIASIQAK----------------------EDLKE.THK.Y..VEI..KSA...VIG..GKA...........C.....V.KSGKPLH....K......S..I......HG
AtCYP20-1 -------MASS----------------VTLLLWSLLLLGTLSAIQAKK-------------------SKENLKEITHK.Y..VEI..KAA..IVMG..GKT....V......C.....I.KNGKALH....S......S..L......HG
AtCYP21-1 MRREISFLLQPRCLL------------LLVALTIFLVFALFNTGKDEEK------------------QVIEDHEITNR..L.VDI..QR...IVIG.YGTV....V......C.....KTSSGKPLH...TP.....S..VI....IIHG
AtCYP21-2 -------MAITATR-----------LVSLTLLWIVVLFVTLALIQIKLTDVADPSVNEKILDAKLNQVGEDLEG.THK.Y...QIN.S.A..ILIG..GNI.........S.C.....V.NMGKPLYF...S.........I.......G
AtCYP23-1 -----MGITRNLILG-----------------LACLAFVSIAKA-----------------------LPHEPELGSAR.V.QTSY-----.DIE.GFYPTVA...VDHIFK.VRLG----------G.NTNH.F.VDK..VA.VA.VASG
OsCYP22-2 -------MAARETSR-----------HASLCLWLALVAATLSLAQAVES-------------------EAELTK.TTK.....TIN.K.A..IVMG..GNT..........IC.....L.KSGKPLS...TP.........I....TVSG
OsCYP23 -------MLRKVAVA------------FLACAALYLAFAAYS--RRESL------------------GEVRLPA.TNR.YL.VEI..QHI..IVIG.YG.V....VA.....C...E.I.HKGKSLH....R.........I....IV.G
OsCYP24-1 -------MAGSGWRRIPAVRRPPMSRPASVCLWIVLVAATLALAQ-------------------AKKSKADLTE.THK.Y..VEI..K.A...VMG..GKT...........C.....T.KSGKALHF...A......S..I......LG
OsCYP24-2 -------MAGSGWRRIPAVRRPPMSRPASVCLWIVLVAATLALAQVLIH--------------LAKKSKADLTE.THK.Y..VEI..K.A...VMG..GKT...........C.....T.KSGKALHF...A......S..I......LG
OsCYP26-1 ----MAALLRHAAAAA------------AAVVLLIAAAASTDGATSF--------------------YASDPNLGSAR.V.QTTH-----.DIE.GF.PHVA...V.HIYK.VRLG----------C.NTNH.F.VDK..VA.VASVVGG
TaCYP23-1 -------MAMRAWRRSAAARAP-----AHLCLWLALVAATLVLAQ---------------------GKKSNLSE.THK.Y...EI..K.A...VMG..GKA...........C.....M.NSGKPLH....S......S..I......LG
TaCYP24-1 -------MLRKAAVG------------FLACLVLYLAFSSYP--RSQRA------------------AYVQLPA.THR.YL.VEI..QNI..IVIG.YGEV....V......C.....D.PKGKPLH...TP.........I....IV.G
TaCYP26-2 ----MAGLLRHIAAA----------------LLFLLTAASTAGASTY--------------------YASDPNLGSAR.V.QLNH-----.DIE.GF.PHVA...V.HIFK.VQLG----------C.NTNH.F.VDK..VA.VA.VMGG
Maize -------METSGWRRRAAARAS--WSPATVYLWLALAAAALTLAQ----------------------AKKDLTE.THK.Y..VEI..K.A..IVMG..GKT...........C.....I.KSGKALH....T......S..L......LG
160 170 180 190 200 210 220 230 240 250 260 270 280
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .
hCypA N----GTGGKSIYGEKFEDENFILKHTGPGILSMANA-GPNTNGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMER-FGSRNGK---TSKKITIADCGQLE---------------------------------
AtCYP19-4 .----.M..E....Q..A....K.......V.....S-.ED........T.VT.S....R.......VQ..DV.YKI.A-E.KQS.T---PKS.VV...S.E.PL--------------------------------
AtCYP20-1 .----.M..E.......A....K.......F......-.QD........T.VT.S....R.......VT..DV.YKV.A-E.NQS.T---PKS.VV.V.S.E.PL--------------------------------
AtCYP21-1 D----.KSSD....GT.P....KIQ.SHA.MVA...T-..DS.......T.V.AS..E.E...L...IQ..DN.F.I.GGA.TYS..---PR..VV...S.EIPKDKWDEER-------------------------
AtCYP21-2 D----.R..E....D..A....K.......F.....S-..DS.......T.VT.S....H.......LS..EV.RKI.A-Q.QDS.V---PKANVI.FAS.EVSL--------------------------------
AtCYP23-1 RSAPMNEEQRKEAEK.IVG.FSDV..VR-.T...GRYDD..SAQ.S.SMLLGNAPH..RQYA.....TK.DETLSKL.EVPTR.E.IFVMPTER...LSTYYYDTKMESCEEERSVLKRRLQASFVEVERQRMKCFP
OsCYP22-2 .----...CD....GM.P....KIN.SA..L.....Y-AKD........T.V.LTR...........LS..DV.YKI.A-E..QS.T---PRS.VL.S.S.E.K---------------------------------
OsCYP23 D----.K.SE....GT.P.....V...H..VIA...S-..DS.....Y.T.I..S....E.....R.IQ..DY.Y.I.GGA.TY...---PR..VV.T.S.EIPKEKWAEEV-------------------------
OsCYP24-1 D----.R..E....T..A....KI......L......-.RD........T.VT.S............LS..DV.YKI.A-E.QQS.S---PKS.VV...S.E.PM--------------------------------
OsCYP24-2 D----.R..E....T..A....KI......L......-.RD........T.VT.S............LS..DV.YKI.A-E.QQS.S---PKS.VV...S.E.PM--------------------------------
OsCYP26-1 RTAPMNDEQQEEAEKSVVG.FSTV..VR-.....GRHSD..SG..S.S.LLGDAPH...QYA...RLTK.DDTLRKL.QLPTR.E.IFVMPIER.S.LSTYYYDVDLESCEAEKSILRRRLSESASEVERWRRKCFA
TaCYP23-1 D----.R..E....T..A....K.......Y......-.RD........T.VT.S............LS..DV.YKV.A-E.KQ..T---PKSIVV...S.EVPL--------------------------------
TaCYP24-1 D----.KARE....GT.P....SV...H..VVA...S-.TDS.....Y.T.I..G....E.....R.IQ..DY.Y.I.GGA.TY...---PR..VL.T.S.EIPKEKWGEEAD------------------------
TaCYP26-2 RKAPMNKEQEQQAEKSIVG.FSTV..VR-.....GRYSD.DSAS.S.S.LLGDAPH...QYA...R.TK.DDTLRKL..LPTHKE.IFVMPIER.E.LSTYYYDIDVESCEAEKSILKRRLSESASEVERWRRKCFA
Maize D----.R..E....M..A....KI......L......-.RD........T.VT.S............LS..DV.YKV.A-E.RQS.Q---PKS.VV...S.E.PM--------------------------------
ER-targeting sequence
7 aa+ + + + * * * *
C#
- * - ++ * * *
- - + + * *
+ + * * #*
+ #
ER-localization signal
βI βII αI αII βIII βIV
βV βVI βVII αIII βVIII
151
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP20-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCYP26-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP27 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP20-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP25 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCYP26-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP31 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP26-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP38 -----------------------------------MAAAFASLPTFSVVNSSRFPRRRIGFSCSKKPLEVRCSSGNTRYTKQRGAFTSLKECAISLALSVGLMVSVPSIALPPNAHAVANPVIPDVSVLISGPPIKDPEALLRYALPIDN
OsCYP47 -----------------------------------MAAALAFPTCCCCRRPSLRPSAGRRGRRPVARCALPSSEKNS---------FSWKEYAISVALSAGLITGAPTLGWPAHASPLE-PVIPDVSVLISGPPIKDPGALLRYALPIDN
TaCYP46-2 -----------------------------------MAAALAS--SRCCRRPSLLTIDRRRS--SVARCALSGGKGNS---------FSWKECAISVALSVGLITVPPTFGWSAHAYPLE-PVIPDISVLISGPPIKDPGALLRYALPIDN
AtCYP37 ---MASPLSSSTVVSHRLFFLHPSPLNRKFLFVKPKLPFNRTNSGDFRMRLHSTSSKTGTKELIHSCNSSIDSKLNTFEAGSKNLEKLVATILIFVQVWSPLPLFGLDSAYISPAEAVLYSPDTKVPRTGELALRRAIPANPSMKIIQAS
OsCYP50 MASRAAAAMVLAGALPVFPGRPLVAAGARCCDGGIRGRVSCSSHRRSDHPSCAAEVSTRWLLVSPHPNGFQSCSVRFSLIVWLSGLVQEGGVVELLKGAVAALAVIAQISVSLPADAILYSPDTNVPRTGELALRRAIPANPNMKTIQES
OsCYP46 MASRAAAAMVLAGALPVFPGRPLVAAGARCCDGGIRGRVSCSSHRRSDHPSCAAE---------------------------------EGGVVELLKGAVAALAVIAQISVSLPADAILYSPDTNVPRTGELALRRAIPANPNMKTIQES
TaCYP36 ----------------------------------------------------------------------------------------------------------------------------------------AIPANPNMKAIQES
TaCYP32 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA -------------------------------------------------------------------------------------------------------------------------MVNPTVFFDIAVD---GEPLGRVSFELFA
AtCYP20-2 -----------------------------------MATLSMTLSNPKSLSAPPRRLSPINTSAFTSTSFRLRTKSSFDSISFSSSTPFSASSLLLHTSYTKRNHRCFSVQSNAEVVTEPQSKITHK.Y...S.GNPV.KLA..IVIG.YG
TaCYP26-3 -----------------------------------------------MAAATSSFATLAIARPAAAGPAAQRALLASKAPSSALSLRGGRIASPALSVSQQSRARFVPSATAEPYAPELQSK.TNK.Y...SIGNPV.KNV..IVIG.YG
OsCYP27 -----------------------------------------MAVATS-FATLAIARPAAERALLASKTPSPLLSIRTGTGTARLPSSAVFGGFTPALSAAHSRARFVSSATADPKEVDLQSKITNK.Y...SIGNPV.KNV...VIG.YG
AtCYP20-3 ------------------------------MASSSSMQMVHTSRSIAQIGFGVKSQLVSANRTTQSVCFGARSSGIALSSRLHYASPIKQFSGVYATTKHQRTACVKSMAAEEEEVIEPQAK.TNK.Y..VEIG---..VA..IVMG..G
OsCYP25 -----------------------------------------------MACRPAVCSPSALAPPRHRLLSLGCARTAAPSGGLPLRLRLRSS-----PAPRGHGADLVVRAAAAEGAVELQAK..SKC...VE.G---...A..IVIG..G
TaCYP26-1 -----------------------------------------------MACRPAVSNPSALTPAPRRCVVLGRS---STRGRVDCSLRSSSAGAVRLGARRAPAAAFVVRAAAAEGDLDLQAK.TSKC...VEIG---..RA.K.VIG..G
OsCYP31 ------------------------------------MQLSPLVSPAMSHQILHTSNPTLPTPPQPHNHHPPVPPPPKLGRRAALAIAAAPAILSATPAPSRAQEAAAAAAAPCIADLPVTAKAFLD.SIGGEPAGRITIG.FGDAAPAG.
AtCYP26-2 -----------------------------MMQPNAKLLSPSAKFLPSPIEPPQHNRRTTVGAPPSLERNCKLSRRNLSKSSLLLLLTTQTTLTPLLDFSKAQADTIANPNLTNCENRIPTKKAFID.SI.GEPIGRIIIG.YGDDVPAGT
AtCYP38 KAIREVQKPLEDITDSLKIAGVKALDSVERNVRQASRTLQQGKSIIVAGFAESKKDHGNEMIEKLEAGMQDMLKIVEDRKRDAVAPKQKEILKYVGGIEEDMVDGFPYEVPEEYRNMPLLKG---RASV.MK.KIKDNPNIEDCV.RIVL
OsCYP47 KAVREVQKPLEDITDSLKIAGVRALDSVERNVRQASRALSNGRNLILGGLAESKRANGEELLDKLAVGLDELQRIVEDRNRDAVAPKQKELLQYVGTVEEDMVDGFPYEVPEEYSSMPLLKG---RATV.MK.KIKDNPN.EDCV.RIVL
TaCYP46-2 KAIREVQKPLEDITDSLKVSGVRALDSVERNVRQASRALTNGRSLILSGLAESKRANGEKTLDKLAVGLEELQRIIEDRNRNAVAPKQKELLNYVGTVEEDMVDGFPYEVPEEYNNMPLLKG---RATV.MT.KIKDNPNVEDCV.RIVL
AtCYP37 LEDISYLLRIPQRKPYGTMESNVKKALKVAIDDKDKILASIPVDLKDKGSELYTTLIDGKGGLQALITSIKKQDPDKVSLGLAASLDTVADLELLQASGLSFLLPQQYLNYPRLAGRGTVEITIEKADGSTFSAEAG.DQRKSATVQIVI
OsCYP50 LEDISYLLRIPQRKPYGSMEGDVKKAMKIAMDNKDAILASIPVELKEKGSKLYTSLLEEKGGLQTLLKYIKENDPDRLSVALASSLDTVAELELLQAPGLSFLLPQQYLEYPRLAGRGVVEFSVEKGDGSTFFPTAG...KSVATIQVVI
OsCYP46 LEDISYLLRIPQRKPYGSMEGDVKKAMKIAMDNKDAILASIPVELKEKGSKLYTSLLEEKGGLQTLLKYIKENDPDRLSVALASSLDTVAELELLQAPGLSFLLPQQYLEYPRLAGRGVVEFSVEKGDGSTFFPTAG...KSVATIQVVI
TaCYP36 LEDISYLLRIPQRKPYGSMESDVKKSMKIAMDNKEAILGSIPAEHKEEGAKLYTSLLEEKGGFQTLLKYIKENDPDKLSIALASSLDIVAELELLQAPGLSFLLPQQYLEYPRLTGRGVVEFTVEKGDGSTFFPTGG...KSVATIQVII
TaCYP32 ------------------MEGDVKKAMQIATENKEAMLASMPAELKEKGSELYTTLLEGKGGLQTLLKYIKDKDNDRLSVALASSLDTIAELELLQAPGLSFLLPKQYLEYPRLTGRAVVEFTVEKGDGSTFFPTAG...KSAATIQVVV
AtCYP28 -----------------------------------------------------MISSSSSSNMASSSILIPPILTRRNLLLSTTIATVTPPPPA---KPPSPDITITDRVFLDFSLCPTYFRSD.SATLSSTTPCSDST.....VLG.YG
OsCYP40 ----------------MVLPSSNTRATSSMAYPVSLSSLHHHPNNHHAFFLPSKTNHDNTHKPIESSRISRRSLIFLPVLPSLLYASSSPALDDANIPSTSAIDTTITDRIFMDFSVCPSYFRSDRTLGAELATCPDS......I.G.YG
OsCYP28 ----------------MVLPSSNTRATSSMAYPVSLSSLHHHPNNHHAFFLPSKTNHDNTHKPIESSRISRRSLIFLPVLPSLLYASSSPALDDANIPSTSAIDTTITDRIFMDFSVCPSYFRSDRTLGAELATCPDS......I.G.YG
Chloroplast/thylakoid targeting signal
Chloroplast/thylakoid targeting signal
Chloroplast/thylakoid targeting signal
Leu-zipperβI βII
D
152
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
....|....|....|....|....|....|....|....|.. ..|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
hCypA D--KVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTR-HNGTGGKSIYG------------------------EKFEDENFILKHTGPGILSMANAGPNTNGS---QFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFG
AtCYP20-2 .--D..Q.V......C...........T...V.RD..I.....EK-G.......V..------------------------RT.K....K.S.V...V.............---......I..S....R.....Q.I...EV.KLI.EQE
TaCYP26-3 .--D..Q.V......C...........S...V.KD..I.....DK-G..........------------------------RT.K....Q.V.....V.............---......V..P....R.....Q.L...D..RTI.SSE
OsCYP27 .--D..Q........C...........S...V.KD..I.....DK-G..........------------------------RT.K....K.V.....VV............---......V..P....R.....Q.I...D..KMI.SQE
AtCYP20-3 E--V....V......C....KY.....S.....KD..I......E-G.....I....------------------------A.......T.....................---......V..S...N......Q.I...KL.RTL.SQE
OsCYP25 E--V....VD.....C..D..Y....CS.....KD..I.....QN-N.....R....------------------------.C.D....T.......V........D....---......V..P...NR.....H.L...DV.KNL.SQE
TaCYP26-1 E--V..R.VD.....C..D..Y....CS.....KD..I.....QN-N.....R....------------------------.C.D....T...V...V........D....---......V..P...DR.....H.L...DV.REL.SQE
OsCYP31 SRFLSLV.GVG---YRRK.FVKIVP.YVQ.GGVVSYPAIPAVTE.LAAEM.AVRAQCGGERSPHAAAGAVSIVVRDPSLPPP.PKLVARGG.LEVDQEQVGVVPNGTEFVITTGDAPELD.SALVVGRVV-DGMD.LGKIAA.PTVKDN-
AtCYP26-2 ARFSSIVSGKAGITYRRKDFVKIMP.YVQ.GG.RSYGVDAERA.AAVGSLQNLIEEWERGKRGEICNVNKAGSVGIVVRDPS.PPPKTKLVARN.KLVVEEEVIAVGP..TEFVITAVDSPEL.DSVLVIGKVL-EGM.VVEKMREVKTV
AtCYP38 .GYNA.V..G..VD.VERH---F.D.MEIQ.SD-..VV.T..PEGPAE.FIDP.TEKTRTVPLEIMVTGEKTPFYGSTLEELGLYKAQVVIPFNAF.TMA..REEFENDSGSSQV.--------..LKESELTPSNSNILDGRY.VFGYV
OsCYP47 .GYNA.V..G..LD.VERK---F.D.MEIQ.AD-..VV.T..PEGPAE.FIDP.TGKVRTIPLELMVDGDKAPVYGETLEELGRYKAQTK.PFNAF.TMA..RDEFDD.SASSQI.--------..LKESELTPSNANILDGRY.VFGYV
TaCYP46-2 .GYNA.V.SG..VD.VERK---F.D.MEIQ.AD-..VV.T..PEGPAE.FIDP.TGKSRTIPLEIMVDGDKAPIYGETLEELGLYKAQTK.PFNAF.TMA..REEFDD.SASSQV.--------..LKESELTPSNSNILDGRYSVFGYV
AtCYP37 .GYSA.L..G..AK.V.SG---A.D.AKLNTVNQAVITED.SGKVESVSVPLEVMPS-------------GQFEPLYRTPLSVQDG.LPV.PLSVY.AVA..HSENSEEY.SPY...FYLYDKRNSGLGGLS.DEGQFSVFGYTI-AGKD
OsCYP50 .GYSA.L..G..AK.VLDG---A.D.IKLKCASQAIIADNENGK-KGYTVPLEVMPA-------------GQFEPLYRTPLSIQDG.LPV.PMSVY.AVA..HSVDSDEY.SPS...FYLYDKRNSGLGGIS.DEGQFSVFGYTT-DGRE
OsCYP46 .GYSA.L..G..AK.VLDG---A.D.IKLKCASQAIIADNENGK-KGYTVPLEVMPA-------------GQFEPLYRTPLSIQDG.LPV.PMSVY.AVA..HSVDSDEY.SPS...FYLYDKRNSGLGGIS.DEGQFSVFGYTT-DGRE
TaCYP36 .GYSA.L..G..AKMVLDG---A.D.VTLKCASQAIIADNETGK-NGYTVPLEVKPA-------------GQFEPLYRTPLSIQDG.LPV.PMSVY.AVA..HSVDSDEY.SPT...FYLYDKRNSGLGGIS.DEGQFSVFGYTT-DGRD
TaCYP32 .GYSA.L..G.IAK.VLDG---A.D.ATLKSVSQAIIVDSETGK-KGYTLPLEVMPA-------------GQFEPLYRSPLSIQDG.LPV.PMSVY.SVA.SHSEDSDEY.SPT...FYLYDKRNSGLGGIS.EEGQFSVFGYAT-DGRD
AtCYP28 R--H..I.VST.KRMC.SS-STS..NTPV.K.F..QYFLA.RQGG-GRRDTAEVG.SLRDLPRNTDVVNSKAFLLPHAR-AGVVSLCLSENDDDDDIR.DPDYRNVEFLITTGPGPSPQLDGGNIVF.TVLEGLD.VTSISSIPTYKPSE
OsCYP40 R--LL.L.TA..K.AC.SA---A.R.TLV.KLLQ.QFFVA.RQGP--RRDR.EVQPPT-GLVRNAETIDPKAFELKHAR-PGTLSLCLGQNDDDDDIK.NPNYHNVEFLVTTGPGPCPELDGQNIVF.TVLEGMN.ITSIATIPTYKPAE
OsCYP28 R--LL.L.TA..K.AC.SA---A.R.TLV.KLLQ.QFFVA.RQGP--RRDR.EVQPPT-GLVRNAETIDPKAFELKHAR-PGTLSLCLGQNDDDDDIK.NPNYHNVEFLVTTGPGPCPELDGQNIVF.TVLEG-----------------
460 470 480 490 500
....|....|....|....|....|....|....|....|.. ..|....|...
hCypA SRNG-KTSKKITIADCGQLE---------------------------------
AtCYP20-2 TDR.DRPR..VV.......PMSEA-----------------------------
TaCYP26-3 TDR.DRPK..VV.SE..E.PVV-------------------------------
OsCYP27 TDR.DRPK..VV.SE..E.PVV-------------------------------
AtCYP20-3 T.AFDVPK.GCR.YA..E.PLDA------------------------------
OsCYP25 TSRSDIPKQPCR.VN..E.PVDG------------------------------
TaCYP26-1 TSRSDIPKQPCR.V...E.PLDG------------------------------
OsCYP31 TASPYFRVA.LIGDKRAVVAERGFNRPYT-KILITNCGVIEQQQEQ-------
AtCYP26-2 RD.-TSSPYFRVAKVI.DKRAVVAERGFNRPYS-KVVVTNCGLIESQTL----
AtCYP38 TD.EDFLADLKVGDVIESIQVVSGLENLANPSYKIAG----------------
OsCYP47 TE.EDYLADLKVGDVIESIQVVSGLDNLANPSYKIVG----------------
TaCYP46-2 TE.EDFLADLKVGDVIESIQVVSGLDNLVNPSYKIVG----------------
AtCYP37 ILGQI..GDI.KS.KLIEGQDRLSLPVQNNNINEST-----------------
OsCYP50 VLSQI..GDI.RS.KLV.GRERLVLPPEAP---AES-----------------
OsCYP46 VLSQI..GDI.RS.KLV.GRERLVLPPEAPAES--------------------
TaCYP36 VLSQI..GD..RS.KLV.GRERLVLPSIAP---EES-----------------
TaCYP32 VLSQI.AGD..RS.KLI.GRERLVLPAAASAPAPADPTPAPADPTLAPAES--
AtCYP28 NIKQFNDFAEFLGDERA.NARSLWNRPLK-TVFISGCGELKVTNPSLSPTLP-
OsCYP40 RIRFFNDFAQLIGDERA.TARALWDRPLK-TVYISDCGELKVTKPSLSPPSLP
OsCYP28 -----------------------------------------------------
+ + + + * * * *
# - * -+ + * **
- -+ + * *
+ + * * # * +
#
αI αII βIII βIV βV βVI βVII αIII
βVIII
153
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGY--------KGSCFHRIIPG-FMCQGGDFTRHNGTGGKSIYG--------------------------------EKF-EDE-NFILKHTGPGILSMA
AtCYP59 --------------MSVLIVTS..DIVID.HS..C.L.CK..LK.CKIKYY-----------N.CL..TVQKD-.TA.T..P.-GT.A..D.-------------------------IYKFLYGEQ-AR.YK..IHLD...SKT.TVA..
OsCYP63 --------------MSVLIVTSV.DIEVD.HT.MC.L.TK..LK.CK.NE.LVA--------G.LA.RKQLS.Q.WRCRAGA.-GS.FDV.GMSFPGYSFFLVFLSLHYFVHIQNYLFSRFLSGAMATR.FD..IHPE.R.SKM.TIA..
OsCYP65 --------------MSVLIVTSV.DIEVD.HT.MC.L.TK..LK.CKMKYY-----------N.CL..KVEK-D.LA.T..P.-GT.A..D-------------------------SVYKFLYGDQA-R.FD..IRPD.R.SKK.TIA..
AtCYP63 ---MTKKK..N..L.VSIG.D.VQ.IVI.....V...........C...A.V.KSTGKPLHF...S...V.K.-..A.....SNG.....E....--------------------------------G..-S..-..R.D.D.A.V....
OsCYP70 MPKAKK--..H......IG.RAAE.IT......V....T......C...R.L.VSTQKPLYF..TNM...LK.-..A.....S.GD.R..E....--------------------------------A..-K..-..K...DQ..V....
TaCYP70 MPKFKKNL..Q..LE.SI..R.AE.IT.Q....V...........C.....L.ESTKKPLYF..THI......-..A.A...SGGD.R..E....--------------------------------G..-P..-..K...DQ.......
AtCYP95 ---MAKKK..Q..M.VSI..D.AETMV....PEVA...S......C.....I.PRSGKPLHY...F....MK.-SSA.A...VNR...A.E...A--------------------------------G..-P..-SPK.R.EET.L...S
OsCYP49 ---MAKKK..I..M.VSIGD..DE.MV......VA.R........C...M.I.QTSKKPLYY...L...V.K.-..A.....SNGD.S..E....--------------------------------GT.-...-..V.R.DER.L....
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA NAGPNTNGSQFFICTAKT-EWLDGKHVVFGKVKEGMNIVEAMERFGSRNG-KTSKKITIADCGQLE------------------------------------------------------------------------------------
AtCYP59 SG.E.L.A...YFTLRDDLDY.....T...QIA..FDTLTRINEAYVDPKNRPY.N.R.KHTHI.DDPFDDPPQLAEMMPDASPEGKPKEEVKDDVRLEDDWVPMDEELGAQELEEVIREKAAHSSAVVLESIGDIPEAEVKPPDNVLFV
OsCYP63 S..E.C.A...Y.TLRDGVDY..D..T...M.A..FDTITKINETYVDDKGRPF.D.R.RHTYV.DDPFDDPPQLSKLIPENSPVGKPQDEIAEER-LEDNWVPPDETVAPEELEDTIRSKEAHTNAVILQSLGDIPDAEIKPQDNVLFV
OsCYP65 S..E.C.A...Y.TLRDDVDY..D..T...M.A..FDTLTKISETYVDDKGRPF.D.R.KHTYV.DDPFDDPPQLSELIPENSPVGKPQDEIAEER-LEDSWVPMDEMVAPEELEEMIRSKEAHTNAVILESVGDIPDAEIKPPDNVLFV
AtCYP63 .C...........LFKRQ-PH...........V...AVIKK..LV.TSD.-.PTSPVK.I...ETSQIRAHDAAEREKGKSKKSNKNFSPGDVSDREAKETRKKESNEKRIKRKRRYSSSDSYSSSSDSDSDSESEAYSSSSYESSSSSD
OsCYP70 ....DS.......TFMP.-PH...........VT..PLLKKL.AV..DT.-.PTCEVK.V...EVSDSQNQLKGEKEKKLRRTEDNSAAEKRVKTQKPPTHDKQKKKRKHYSSDSYSSDYSDTQSSDSGSESESYSSSSLDTSSSSDHRH
TaCYP70 .S.E.........TFKAV-PH...........LN.KALLKKL.AL..ES.-.PTCPVK.V...EASNIDTQNQLHGEKEKKLKRAVEYNNSDAGGRVKTKKTSSVDKRRKRRKNYSSDSYSSETSDSQSYSSDSGSESQSYSSASSDTSS
AtCYP95 I.DRDKF..H.H.TFRPN-QQ..RNN.....LIQ.KE.LKKI..V.DEE.-.PTVSVK.IR..EYSGDKKKSDGKKNGKHKKSLRVRRKKRRRHSSSESESSSDSETDSSESDSESDSDLSSPSFLSSSSHERQKKRKRSSKKDKHRRSK
OsCYP49 .............TFKHN-SR..R.ST....LIL.NDVLKRI.YVDVHGAGS.PVVPVRIVDCGELVDGKCLGSITVENDKKRSVKSKLSKDESSDEENNEGKRKRHHKKSSRRRRKKKRYSSSESESSSESESELSDSDSESDTCSSDS
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP59 CKLNPVTEDEDLHTIFSRFGTVVSADVIRDFKTGDSLCYAFIEFENKESCEQAYFKMDNALIDDRRIHVDFSQSVSKLWSQFRQKDSQKGKGNGCFKCGSTDHIAKDCVGGPSSK-----FIVKDQNRQHGGGE--GYEMVFEGDVHETP
OsCYP63 RELN---KDEDLYTIFSHFGSVTSAEIIRDYKTGDSLCFAFIEFEKKEACERAFFMMDNCLIDDRRIRVDFSQSVSKQWRQFRQSKSNANK-DGCFKCGALDLIARDCDQRAEQKNKGPNYILKDENTQRSGNKRRSYDLVFE-DGENYN
OsCYP65 CKLNPVTQDEDLYTIFSRFGTVTSAEIIRDYKTGDSLCYAFIEFETKEACERAFFKMDNCLIDDRRIHVDFSQSVSKLWGQFRQSKRNANK-DGCFKCGAPDHIAKDCDQGTEQKNKGPSYVLKDENTQRGGNNRRSYDLVFDEDGENYT
AtCYP63 GKHRKRKSTTRHKGRRGERKSKGRSGKKKARPDRKPSTNSSSDTESSSSSDDEKVGHKAIKSVKVDNADQHANLDDSVKSRSRSPIRRRNQNSRSKSPSRSPVRVLGNGNRSPSRSPVRDLGNGSRSPREKPTEETVGKSFRSPSPSGVP
OsCYP70 KRRKSSKKDKHRSAKGKSKHKKTKRKSRGTKRKSKRSYRSSSDDSDSSKTGGSSSDSESEGRRTTRTKHSSKKDPDNTKTISLEKDSTLEDADKGKQTATLDN-ISNEGSKPSNTDGNGAGIRDDPGARARSSPIRADASLTKVDGNNGA
TaCYP70 SSDHRHKRRKGSKKVKRKPAKRKSSHKKSKSKSRGTKRSKRSYGSSSDASKSSSTSSDNESAGRRTKHPLKKDQENTKIINLEIGKSLEYADKGKQTVAGSKPLHKDESWADDRVGTQNSEDRSSKFRDDTNPIRADTTLSKADGNITAV
AtCYP95 QRDKRHEKKRSMRDKRPKRKSRRSPDSLEDSNSGSEASLSDVNVEIGAKKRKHRVSRRTGNSAPAVEKEAESLHQGKRKGPDLLENRGLRSNGISDAASEQISDRQPDIVDDHPSKSRSRSLSPKRTVSKSTSVSPRRSQSKSPSSSPRW
OsCYP49 SDLSSSSDDRRRRRKRHSKKDKHKRGKRKRDRRRERKRRKRDRKSKQKSKRMLESDSETGNVSDSSLEDDKSKRHHRGRKSKASSQVSGENHTALAALKDAASTQQKSATPRSLAQEDKSPKENGDTRTNGVTESKTERNADIALTSNRS
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP59 KHN---SHERERSEKIQ-RRS---PHGNGE---GKRQHRDERD-----DGRRQHDREDARELERKHRERKERESREDE---DRRRRRRREESRDKESRRERDEDDHRSHRDYK-ERRRERDDRHGREARHERRDR---------------
OsCYP63 GQQDLRSADRRKIHKIDDRRSGLPPRGDRDRISRERTHIDEND----KEGNRDRGNQKHEDYNRYCKPGERSSSRHDDRGYSKHESRSKYRDGDDDYRRQSGG-SRYGRDKCDGERRYRGDDDHGRSNRHTR------------------
OsCYP65 DQQDPGSTGRRKIQRTDDRKSGLPPRGDHDRISRERTHSDENGREGGKEGDRDRGIRKHEDYHRYNKSGERSSSRYDDRGYSKHESRGKYRDGNDDYRRQPGGGSRYGRDKYEGERRYREDDGHGRSDRHKRDESDNRKRSPDTGKHRRE
AtCYP63 KRIRKGRGFTERYSFARKYHTPSPERSPPRHWPDRRNFQDRNRDRYPSNRSYSERSPRGRFRSPPRRRSPPRYNRRRRSTSRSPDGYRRRLRDGSRSQSPRHRSRSQSPRKRQPISQDLKSRLGPQRSPIRGGRTSPAESLSPSHSPSPP
OsCYP70 DTAEAGISRAEPVPTNGKDLAMGSTDNGQPQRVRKGRGFTQQYAFARRYRTPSPERSPVRSRYNDGRNDRWNHFNRYGRNGPYGARSPVRRYRGSPRASSPSRYPRRDRSRSRSRSPLRYRERGGYRRPSPRHSRSRSPAEHQRRDVRNR
TaCYP70 AAGTGISEAGAERNPLSNKPVPTNGQDLAMGSTEDGRVRKGRGFTQKYSFARRYRTPSPECSHVRSSYYGGRNDRWNNFNRYGRNGLYGARSPVRRYQGSPRASSSLRYTRRDRSRSRSRSPVRRRDGGGRHRRPSPRRSHSPAEQHKRD
AtCYP95 NGGRSPAKGSRQVKNLTNSRRESPGSEEKGRHVRRSPTKSVSRSPVRVKKERDISRSPSKSLSRSPLRSPKRVISRSPVRGRIARSPSRSPVRSASRGSLGRGPLRRSSRRSPSRSPVRSSRRSLSRSPIQLSRRSLSRSPTRLSRRSLS
OsCYP49 KSRFPIFLHTSIILCAYCVHEQCFI-----------------------------------------------------------------------------------------------------------------------------
E 8 aa+ + + + * * * * # - * -
+*
+ * *
+ + * *
+ **
#+ *
+ #
βI βII αI αII βIII βIV βV
βVI βVII αIII βVIII
RRM
RRM
Zinc-Finger
Arg/Lys rich domains
Arg/Lys rich domains
- -
154
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ------------------------------------------------------------------------------------------------------------------------------------------------------
AtCYP59 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP63 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP65 DGGHRETSKHRERRQRDDR-----------------------------------------------------------------------------------------------------------------------------------
AtCYP63 GKRGLVSYAD--------------------------------------------------------------------------------------------------------------------------------------------
OsCYP70 LRSGRDGGGPDHRNSSPPVNRGRSRSRSKSRDPSKSRSPDAPAKKGSSKYNRRRSSSSRSSSPAGSKGLVSY------------------------------------------------------------------------------
TaCYP70 AAYRPRSGRGGGGGPSAANRGRSRSRSKNRDASRSRSPNAAPAKRLSSKYNRRRSSSSRSSSPSGSKGGLVSY-----------------------------------------------------------------------------
AtCYP95 RSPIRSPRKSVSRSPVRSSRKSVSRSPVRSSRRRISRSPVRSSRKSVSRSPIRLSRRSISRSPIRLSRRSISRSPVRGRRRISRSPVPARRRSVRPRSPPPDRRRSLSRSASPNGRIRRGRGFSQRFSYARRYRTSPSPDRSPYRFSDRS
OsCYP49 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .
hCypA -------------------------------------------------------------------------------------------------------------------------------
AtCYP59 -------------------------------------------------------------------------------------------------------------------------------
OsCYP63 -------------------------------------------------------------------------------------------------------------------------------
OsCYP65 -------------------------------------------------------------------------------------------------------------------------------
AtCYP63 -------------------------------------------------------------------------------------------------------------------------------
OsCYP70 -------------------------------------------------------------------------------------------------------------------------------
TaCYP70 -------------------------------------------------------------------------------------------------------------------------------
AtCYP95 DRDRFRSRRRFSPSRFRSPLRGRTPPRYRRRSRSVSPGLCYRNRRYSRSPIRSRSPPYRKRRSPSASHSLSPSRSRSRSKSYSKSPIGTGKARSVSRSPSKARSPSKSDSTSSDNSPGGKKGLVAYD
OsCYP49 -------------------------------------------------------------------------------------------------------------------------------
Arg/Lys rich domains
Arg/Lys rich domains
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA ----------------------------------------------------------------------MVNPTVFFDIAVD---GEPLGRVSFELFADKVPKTAENFRALSTGEKGFGY-KGSCFHRIIPGFMCQGGDFTRHNGTGGK
AtCYP21-3 MAKIKPQALLQQSKKKKGPSRISITNIVIYTLAVLLLVFVLFSAYRRWTHRS-EIPTHNGRS-VLEDAAFP-G-MKNV.LPRFATLDTGK.S.TI...K.TA.NVVDQ.MKFCQDG----.F..FL.S.VVKH.VI.A..SAEFDAVKDW
AtCYP21-4 MAKIKPQALLNQSKKKKGPSRISISTIIVCNLVVAVVILSLVTTYRHWSQRSRNTIEHETRSQRFEDTNTASG-QKTY.LPGFADINTSK.LITV...KEGS.EVVDK.LD.CQKD----HF..MP.Q.V.KNYLV.A.HSPSSIPVEEW
OsCYP26-2 MARIKPKQLLIQSKTKKAPTRISYSTIVTWNLIVILVALSLYATYRHWHHRPMLETEMDLPRAEHVGRSEDST--KTSRPSYAVIDTA-K.SITI.IYK.ASADVVDR.VS.CKSN----HF..MP.RHV.KN.VI.....DFNGAAQEW
TaCYP26-4 MXRLKXKQLXIQSKXKKGPSXISYXSIIXWKXXVXLVVLSXYATYRHWHHRPAFEAEMDLPRAENVERSEDST--KISTPSYVIMDTL-K.SITI.ISK.ASAGVVDR.IN.CKSD----.F..MP.RHV.KNSVIH....DLNGAAHEW
TaCYP16-1 MARIKPKQLLIQSKTKKGPSRISYSSIITWNLIVVLVVLSLYATYRHWHHRPAFEAEMDLPRAENIERSEDST--XISTPSYVIMDTF-E.SITI.ISQ.ASAGVVDR.INFMQSD----.F..MP.RHV.KNWESM..ILI--------
TaCYP23-2 MARIKPKQLLIQSKTKKGPSRISYSSIITWNLIVVLVVLSLYATYRHWHHRPAFEAEMDLPRAENIERSEDST--KISTPSYVIMDTL-K.SITI.ISK.ASAGVVDR.IN.CKSD----.F..MP.RHV.KNLVIY....DLNGAAHEW
160 170 180 190 200 210 220 230 240
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hCypA SIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKT-EWLDGKHVVFGKVKEGMNIVEAMERFGSRNG-KTSKKITIADCGQLE----
AtCYP21-3 ALD---RKNI-DTSLKHEE-FMVGTPKAK.EQ.GFE.FIVSAQIKD.NE.LT...R.SK.QDV.QEI.EVETDDQYQPKSP.E.MSVTL.QDM--
AtCYP21-4 TAK.KLRGRL-H.GPKHEA-FMLGTPKNKGN.KDFELLI.TAPIPD.NDQLI...R.LK.EDV.QEI.EVDTDEHFQPKSP.G.TGVVLKLET--
OsCYP26-2 ILKAKASGEN-ALSPKHEA-FMIGTTKN.-N.KGFDLFI.TAPIPD.ND.L....Q.IN.QD..QEI.EVDTDEHYQPKTP.G.LNITLKQQALS
TaCYP26-4 ILKAKASGEN-ALSPKHEA-FMIGTTRS.-NSKGFDLFI.TAPIPD.ND.I.L..R.IK.ED..QEI.EIDTDEHYQPKTP.G.INISLIQEL--
TaCYP16-1 -----------------------------------------------------------------------------------------------
TaCYP23-2 ILKAKASGEN-ALSPKHEA-FMIGTTRS.-NSKGFDLFI.TAPIPD.ND.I.L..X.IK.ED..QGD----------------------------
Mitochondria targeting sequence+ + + + * * * *F # - * -
+ + * **
+ + * *
+ ** #
+ * + #
βI βII αI αII βIII βIV
βV βVI βVII αIII βVIII
4 aa
- -
Fig 5.5 Alignment of putative amino acid sequences of rice and wheat Cyps with reported Cyps in Arabidopsis and human CypA. A: cytosolic single domain Cyps; B: cytosolic multi-domain Cyps; C: ER Cyps; D: chloroplast Cyps; E: nuclear Cyps; F: mitochondrial Cyps. Dots indicate amino acids identical to human CypA. Secondary structure was generated based on hCypA. Asterisks (*) above the lines indicate residues involved in CsA contact; plus (
+) above the lines indicates residues involved in enzymes activity;
Number (
#) above the lines calcineurin binding residues; minus (
−) above the
lines indicates N-linked and O-linked glycosylation sites. Framed motifs are ER targeting signal, chloroplast/thylakoid lumen targeting signal, mitochondrial targeting signal, Arg/Lys rich domain, TPR: tetratricopeptide repeat, WD: 40-WD repeat, RRM: RNA recognition motif, leu-zipper; Zinc-finger.
155
OsCYP18-2
OsCYP20
OsCYP19-3
TaCYP18-1
TaCYP17
AtCYP18-2
TaCYP16-2
AtCYP18-1
OsCYP18-1
TaCYP31-1
TaCYP21
AtCYP22-1
OsCYP22-1
TaCYP24-2
OsCYP19-2
TaCYP19
AtCYP19-3
hCypA
OsCYP18-3
TaCYP18-2
AtCYP18-3
AtCYP19-2
AtCYP19-1
AtCYP18-4
AtCYP26-1
OsCYP19-1
OsCYP16
74
100
100
100
37
100
96
100
100
72
62
57
94
99
43
32
77
35
85
54
71
52
46
60
A
B OsCYP43
OsCYP44
TaCYP46-1
AtCYP40
hCypA
AtCYP65
OsCYP51
AtCYP71
OsCYP58
OsCYP73
AtCYP57
OsCYP57
TaCYP31-299
100
100
100
100
62
100
100
93
62
156
OsCYP24-1
OsCYP24-2
Maize
TaCYP23-1
AtCYP19-4
AtCYP20-1
AtCYP21-2
OsCYP22-2
hCypA
AtCYP21-1
OsCYP23
TaCYP24-1
TaCYP26-2
AtCYP23-1
OsCYP26-166
100
99
92
76
86
100
85
84
98
75
C
OsCYP50
OsCYP46
TaCYP36
TaCYP32
A tCYP37
OsCYP47
A tCYP38
TaCYP46-2
hCypA
TaCYP26-3
OsCYP27
A tCYP20-2
A tCYP20-3
OsCYP25
TaCYP26-1
A tCYP28
OsCYP40
OsCYP28
OsCYP31
A tCYP26-2100
98
100
100
71
94
100
100
71
70
100
100
85
77
91
97
D
OsCYP70
TaCYP70
AtCYP63
hCypA
AtCYP95
OsCYP49
AtCYP59
OsCYP63
OsCYP65100
100
98
31
32
40E
157
TaCYP26-4
TaCYP23-2
TaCYP16-1
OsCYP26-2
AtCYP21-4
AtCYP21-3
hCypA
63
100
56
99F
Fig 5.6 Phylogenetic analysis of Cyps in Arabidopsis, rice, and wheat. A: Cytosolic single domain Cyps; B: Cytosolic multi-domain Cyps; C: ER Cyps; D: Nuclear Cyps; F: mitochondrial Cyps. Trees were generated by Neighbor-Joining of the Bootstrap Test of Phylogeny in MEGA 4.0 program (http://www.megasoftware.net/mega.html), based on the alignments of Cyps by ‘ClustalW Multiple Alignment’ in Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).
5.3.8 Chromosomal localisation of Cyps in rice
The maps of chromosomal locations of Cyp genes in rice were generated from the
Oryza sativa genome view in the NCBI Map viewer database
(http://www.ncbi.nlm.nih.gov) with representative cDNA/ESTs listed in Table 5.1 (Fig
5.8A). Twenty-seven loci, encoding 33 putative Cyps, were scattered on all
chromosomes except 4 and 12. Six loci, encoding seven Cyps, were on chromosome 6,
four loci each were on chromosomes 2 and 8 and three loci each on chromosomes 1 and
7. Two were present on chromosome 3, with genes at each end, and on chromosome 9,
with genes at one end. One each occurred on chromosomes 5, 10 and 11. Some of rice
Cyp genes are closely linked, e.g., the four loci (encoding OsCYP22-2, OsCYP63,
OsCYP65, and OsCYP24-1+OsCYP24-2 encoded by one locus) in ~22 kb on
chromosome 6; three (encoding OsCYP16, OsCYP18-3, OsCYP49) in ~52 kb on
chromosome 2, and two (encoding OsCYP23 and OsCYP19-2) in ~16 kb on
chromosome 9. The distance between two adjacent Cyps varied from 1014 bp
(OsCYP22-2 and OsCYP24-1) to 9.9 Mb (OsCYP47 and OsCYP73).
158
Chr1
OsCYP31
OsCYP57
OsCYP25
Chr2
OsCYP18-3
OsCYP44
OsCYP49
OsCYP16
Chr5
OsCYP27 OsCYP43
OsCYP18-1
Chr6
OsCYP24-1;
OsCYP24-2
OsCYP63
OsCYP65
OsCYP22-2 OsCYP26-2
Chr7
OsCYP46;
OsCYP50
OsCYP70
OsCYP18-2; OsCYP19-3;
OsCYP20Chr8
OsCYP28;
OsCYP40
OsCYP47
OsCYP58;
OsCYP73 Chr10
OsCYP19-1
Chr11
OsCYP26-1
Chr9OsCYP19-2
OsCYP23
OsCYP22-1
Chr3
OsCYP51
Fig 5.7 Likely locations of rice Cyp genes on chromosomes. The map was generated from the Oryza sativa Genome View in the NCBI Map viewer database (http://www.ncbi.nlm.nih.gov) with representative cDNA/ESTs as queries. Frames indicate genes likely to be different splice forms.
5.3.9 Prediction of chromosomal location of Cyps in wheat
A search in the ‘landmark or region’ for each of the 27 Cyp loci on the rice genome
browser (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/) led to identification of
three wheat bin mapped markers (Appendix VII). These were entered into the ‘Mapped
Loci for EST-derived Probes’ query in the GrainGenes structured query language (SQL)
database (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi), leading to the
identification of cDNA probes used for Southern blotting. This showed
KSU027BE590822 had been mapped to wheat chromosome 1 (1AL: 3-0.61-1.00, 1BL:
2-0.69-0.85, 1DL: 2-0.41-1.00) by two probes (5’ probe: BE590822, 3’ probe:
CD453728), NDS021BE406148 to chromosome 7 (7AL: 1-0.39-0.71; 7BL: 7-0.63-
0.78; 7DL: 2-0.61-0.82) by 5’probe (BE406148), UMW213BE497013 to chromosome
3 (3AS: 4-0.45-1.00) (Appendix IV). The sequences of 5’ probes were searched in the
EST database (http://www.ncbi.nlm.nih.gov) and aligned with the corresponding wheat
TAs. The cDNA sequences of BE590822 and BE406148 showed identities of 95.2%,
and 95.7% in respective areas overlapping with TaCYP23-1; and BE497013 aligned
with corresponding sections of TaCYP26-1 (99.8% identity). The maps were then
generated based on deletion breakpoints (http://www.k-
state.edu/wgrc/Germplasm/Deletions ) (Fig 5.8). TaCYP23 was probably located on the
159
long arms of group 1 chromosomes, while it had been experimentally mapped to the
long arms of group 7 chromosomes (see section 4.3.3). TaCYP26-1 was probably
located on short arm of chromosome 3A.
TaCYP23-1
1AL-3:
0.61-1:00
TaCYP23-1
1BL-2: 0.69-0.85
TaCYP23-1
1DL-2: 0.41-1:00
TaCYP26-1 3AS-4: 0.45-1:00
3A
Fig 5.8 Summary of likely locations of wheat Cyps on chromosomes. Diagram of deletion breakpoints from the wheat genetic and genomic resource centre (http://www.k-state.edu/wgrc/Germplasm/Deletions, accessed 06/2009).
160
5.3.10 Preliminary expression analysis of genes encoding putative ER localized
Cyps
Expression of wheat genes TaCYP23-1, TaCYP24-1 and TaCYP26-2 encoding putative
ER localised Cyps was analysed by reverse transcriptase PCR (RT-PCR). First-strand
cDNA synthesised from total RNA from stems, leaves and developing seeds (6, 10, 14,
18, 25 and 30 days after anthesis (DAA)) was amplified separately with primer pairs
WC4/CYPBR1, CYP21-1F2/CYP21-1R and CYP23-1F2/CYP23-1R2 (Table 2.8).
Amplification of gDNA was also conducted to ensure the isolated RNA had no gDNA
contamination. The ACTIN gene was amplified simultaneously from all cDNAs with
the primers ACTIN-F/ACTIN-R as a ‘housekeeping’ control (Fig 5.9). The expression
of genes (TaCYP23-1, TaCYP24-1, TaCYP26-2, ACTIN) in the first set of samples
(panels A, B, C, D) and in the second set of samples (panel E, F, G, H). The sizes of
PCR products were ~500 bp (TaCYP23-1), ~550 bp (TaCYP24-1) and ~450 bp
(TaCYP26-2). The gene TaCYP23-1 obtained from gDNA is ~2000 bp (see section
3.3.2). PCR of cDNA conducted with the ACTIN primers (based on its cDNA;
Accession No. AB181991) was of the expected size (410 bp). The preliminary results
indicate that all the genes encoding putative ER-localised Cyps are expressed in all
tissues. Most gels indicate that the expression in stem and leaf is lower compared to
that in developing seeds. However; the work was only designed as a qualitative test for
confirming previous reports of Cyp expression in developing endosperm (Grimwade et
al., 1996) and was not set up for robust quantitative analysis. The presence of distinct
bands of expected sizes confirms expression in developing seeds; quantitation will need
further work.
Table 5.5 Primers used for expression analysis of ER-localising Cyp genes*
Names of Primer Sequence Expected size (Annealing Temperature)
TaCYP23-1 WC4 5’CGCACAAGGTCTACTTCGA3’ 496 bp (54
oC)
CYPBR1 5’CTGTCGGCAATGACAACC3’
TaCYP24-1
CYP21-1F2 5’GGCCGTCGGGTTCCT3’ 542 bp (52oC)
CYP21-1R 5’CCCCTTCAATGGCGT3’
TaCYP26-2
CYP23-1F2 5’ACGCCTCGGATCCCAACC3’ 447 bp (63oC)
CYP23-1R2 5’GGGTTGGCAGGCGCTCT3’
*Taken from original Table 2.9; provided here for convenience.
161
500bp
100bp
M S L 6 10 14 18 25 30 G
A
M S L 6 10 14 18 25 30 G
600bp
100bp
B
M S L 6 10 14 18 25 30 G
500bp
100bp
C
500bp
100bp
M S L 6 10 14 18 25 30
D
M S L 6 10 14 18 25 30 G
500bp
100bp
E
M S L 6 10 14 18 25 30
600bp
100bp
F M S L 6 10 14 18 25 30
500bp
100bp
G
500bp
100bp
M S L 6 10 14 18 25 30
H Fig 5.9 RT-PCR products amplified from the first-strand cDNA or genomic DNA of various tissues and developing seeds of wheat First set of samples (A, B, C, D), A: TaCYP23-1; B: TaCYP24-1; C: TaCYP26-2; D: actin gene. Second set of samples (E, F, G, H), E: TaCYP23-1; F: TaCYP24-1; G: TaCYP26-2; H: actin. M: GeneRuler
TM DNA ladder; S: stem, L: leaves, G: genomic DNA, 6-30: developing seeds at
different DAA (days after anthesis). The PCR products were separated on 1.0% agarose gels.
162
5.4 Discussion
5.4.1 The Rice and wheat Cyps comprise large and divergent families, with some
duplicated genes
This study has identified 27 loci encoding 33 putative Cyps in rice, including five loci
potentially encoding alternative splice forms. The putative proteins range from 16.2 to
72.9 kDa. The Cyps in rice are located in different cellular locations, as reported for
Arabidopsis (Romano et al., 2004a). Gene structures of Cyps in rice varied from
intronless to 15-exon. Five of the six intronless genes (OsCYP16, OsCYP18-1,
OsCYP18-3, OsCYP19-1, OsCYP19-2) encoded cytosolic Cyps, the same number as in
Arabidopsis (He et al., 2004). OsCYP18-3 and OsCYP19-2 were reported as Cyp2 and
Cyp1 respectively; and Cyp2, with an adenyl rich region at 5’ of the transcript, might be
translated during stress conditions (Buchhoz et al., 1994). OsCYP24-1 (CypB in chapter
3) shows a putative promoter with tissues specific and ER stress responsive elements
(3.3.10). Some rice genes are in close physical vicinity, suggesting a multiple gene
duplication events, leading to tandem copies such as loci for OsCYP22-2 and OsCYP24-
1+OsCYP24-2 which are separated by only 1014 bp, or loci for OsCYP43 and
OsCYP44 on different chromosomes. Twenty-two TAs in wheat were identified to
encode putative Cyps ranging from 15.5 to 70 kDa, also in five cellular locations.
TaCYP18-1 has been reported earlier as TaCYP18-3 on chromosome 6AS (Johnson and
Bhave, 2004b). TaCYP23-1 is the TaCypB-A on 7AL (sections 3.3.5, 4.3.3) and its
promoter from 2n wheat contained tissues specific elements (section 3.3.10). Some of
the Cyps group together and are conserved in comparable areas, suggesting their genes
may be homeoalleles or duplicates.
5.4.2 Single domain cytosolic Cyps
This study identified nine single domain cytosol Cyps in rice and eight in wheat. The
residues R55, F60, H126 in hCypA shown to be essential for PPIase activity (Zydowsky
et al., 1992) were substituted by Q, I or Y respectively, in various combinations in
OsCYP16, OsCYP18-1 and TaCYP16-2 (Tables 5.1, 5.3), suggesting that Q/I/Y may
have roles in PPIase activity, or that these isoforms have no enzymatic roles. Further
studies should be conducted to test the PPIase activity and CsA binding of these Cyps or
their orthogues in Arabidopsis. The residue W121 in hCypA, essential for CsA binding
(Bossard et al., 1991) was replaced by H or S in various rice or wheat isoforms,
163
suggesting the residues H and S may fulfil the role of CsA binding/immunosuppression.
Binding of calcineurin (CN) to the CsA-CypA complex can increase stability and avoid
the proteolytic cleavage of this complex (Hornbogen et al., 1992). The R69 essential
for CN binding (Rascher et al., 1998) was replaced by A or Q, or was lacking in some
isoforms from both plants. The PPIase and/or chaperone activity of such Cyps or their
orthologues (AtCYP18-1, AtCYP18-2 and AtCYP22-1) in Arabidopsis or in other
organisms has not been addressed. Glycosylation seems to have a role in protecting the
protein from digestion by proteinase K (Hirtzlin et al., 1995). N-glycosylation is
associated with N71 and N108 in hCypA; N71 was replaced by T or D in various rice
and wheat Cyps, but N108 was strictly conserved. Likewise, of the O-glycosylation
sites T73 and S110 in hCypA (Pelle et al., 2002), T73 was replaced by H, K or R, while
S110 was conserved in all. Further, the residue G between the N-linked glycosylation
and O-linked glycosylation is conserved in all Cyps except OsCYP16 (Fig 5.5A). The
impact of these partially conserved sites on glycosylation and stability of these Cyps,
and their activities as PPIases and/or chaperones, is unclear.
The insertion between α-helix-II and β-strand-III is a characteristic of plant Cyps (Saito
et al., 1999; Johnson and Bhave, 2004b), its length being variable with 8, 7, 4, 3 or 1 aa.
Also, there is a deletion of 5 aa in this region in some Cyps. OsCYP20 contains a 12 aa
insertion between β-strand-IV and β-strand-V, outside the pocket (β-strand-III to β-
strand-VI) for CsA binding and PPIase activity (Pflugl et al., 1993). However, the 12 aa
deletion of OsCYP20 happens inside the pocket for CsA binding and PPIase activity,
suggesting it may influence its PPIase activity. Further study should assay for the
PPIase activity of OsCYP20 for addressing the influence of deletion.
The 18 kDa Cyp isoforms are reported to regulate K+ channels in guard cell in Vicia
faba (Luan et al., 1993), plant growth in tomato (Oh et al., 2006) and Solanum
sogarandinum (Kielbowicz-Matuk et al., 2007) and stress response induced by
pathogens in potato (Dubery, 2007). The 20 kDa isoforms with unclear cellular location
are reported to be responsible for stress induced by drought in sorghum (Sharma and
Singh 2003; 2006). Their equivalents, i.e., TaCYP18-1, TaCYP18-2 and TaCYP19 in
wheat and OsCYP18-2, OsCYP18-3, OsCYP19-1 in rice) need to be tested for similar
roles.
164
5.4.3 Multi-domain cytosolic Cyps
Six multi-domain cytosolic Cyps in rice and two in wheat have been identified. H126
was replaced with N in OsCYP57 and TaCYP31-2, W121 replaced by H and R69
replaced by A or missing in some isoforms (Tables 5.1, 5.3; Fig 5.5B). AtCYP71 with
a deletion at this position acts as a highly conserved histone remodeling factor involved
in gene silencing (Li et al., 2007), and AtCYP40 (with A) has been recently shown to be
involved in regulation of vegetative phase development (Smith et al., 2009). Thus CN
binding may not influence some activities of Cyps. N71 was replaced by D/S/T in all
these Cyps. AtCYP40 and AtCYP71 with D and AtCYP65 with T at this position do
show PPIase and/or chaperone functions (Li et al., 2007; Wiborg et al., 2008; Smith et
al., 2009); thus these replacements do not appear to affect some roles.
There are 6-8 aa insertions between α-helix-II and β-strand-III, corresponding to the 7
aa insertion characteristic of plant Cyps (see above). Also, 1-2 aa insertions or deletions
exist between β-strand-IV to β-strand-VI, which may not influence the pocket (β-strand-
III to β-strand-VI ) for CsA binding and PPIase activity (Pflugl et al., 1993). N-terminal
variable extensions (19 aa in TaCYP46-1 to 477 aa in OsCYP73) exist in six of the
multi-domain cytosolic Cyps (OsCYP43, OsCYP44, OsCYP51, OsCYP58, OsCYP73,
TaCYP46-1). The WD-40 repeat, a protein-protein interaction unit, consists of about 40
aa with W-D in the centre (reviewed in He et al., 2004). OsCYP73 presents three WD-
40 repeats, similar to its AtCYP71, while OsCYP58 has two, and all the other functional
residues are conserved (Fig 5.5B). Thus OsCYP58 and OsCYP73 may also be involved
in histone remodeling (Li et al., 2007). OsCYP43, OsCYP44, TaCYP46-1 exhibit a
TPR motif shown to interact with heat shock protein (hsp90) complex (Ratajczak et al.,
1993). The human Cyp40 with TRP show chaperone functions (Mok et al., 2006).
OsCYP43, OsCYP44, TaCYP46-1 share the important residues listed in Table 5.4 and
the TRP consensus with their orthologue AtCYP40, thus these isoforms may also
regulate miR156-regulated members responsible for vegetative phase change (Berardini
et al., 2001; Smith et al., 2009).
165
5.4.4 ER Cyps
Five ER-localised Cyps in rice and three in wheat were identified. The various isoforms
show replacements of H126 by Y, W121 by R and H, and/or R69 by S/L/G (Tables 5.1,
5.3; Fig 5.5C). The N71 is replaced by D in five Cyps and R in two; and N108 is
replaced by G or A in one Cyp each. Further studies are required to test the
glycosylation status and/or PPIase activity of the relevant ER isoforms.
The plant-specific seven amino acid insertion between α-helix-II and β-strand-III (Saito
et al., 1999; Johnson and Bhave, 2004b) exists in four rice and two wheat Cyps but is
absent in OsCYP26-1 and TaCYP26-2. A 5-aa deletion between β-strand-I and β-
strand-II, a 4-aa insertion between β-strand-I and β-strand-II, and a 3-aa insertion
between α-helix-III and β-strand-VIII occur in OsCYP26-1 and TaCYP26-2. All these
insertions or deletions are outside of the pocket (β-strands III to β-strand-VI) for CsA
binding and PPIase activity (Pflugl et al., 1993) and this may have no relevance to the
PPIase activity. The N-terminal ER-targeting sequences (Price et al., 1991) exist in all
these Cyps with variable lengths. The AtCyp5 (AtCYP19-4, Romano et al., 2004a) in
Arabidopsis is suggested to have a potential cleavage site for the signal peptide between
A23 and K24 (Saito et al., 1999). The rice and wheat Cyps present the putative
cleavage sites between A39 and K40 (OsCYP24-1; rice CypB in chapter 3), A44 and
K45 (OsCYP24-2) and G34 and K35 (TaCYP23-1; TaCypB7B, chapter 3). C-terminal
ER-localisation residues are present in all the Cyps with different lengths, with the four
residues being similar but not identical to the characteristic KDEL/HDEL/HEEL ER-
retention/retrieval signals (Frigerio and Pelham, 1993; Derkx and Madrid, 2001); 11
residues (OsCYP23 and TaCYP24-1) are similar to the hCypB (Boldbaatar et al., 2008).
The role of the unusually long 36-residue C-terminus of OsCYP26-1 and TaCYP26-2 in
ER retention needs to be confirmed, e.g., by deletion of this sequence in these or their
orthologue AtCYP23-1 and checking their cellular locations.
The orthologues AtCYP19-4 and AtCYP20-1 of TaCYP23-1, OsCYP24-1, OsCYP24-2
can regulate the protein GNOM required for coordination of cell polarity along the
embryo axis (Grebe et al., 2000) and PP2A (protein phosphatase 2A) involved in auxin
transport and growth response (Jackson and Soll, 1999). OsCYP23 and TaCYP24-1
contain an 11-residue C-terminal, similar in length (but not sequence) to the ER-
localisation residues of hCyP-22b/p (NP_000933) that acts as a transcriptional inducer
166
(Rycyzyn and Clevenger, 2002) and plays crucial roles in protecting cells against ER
stress (Kim et al., 2008). Further studies should focus on characterising the genes
encoding the newly identified ER isoforms TaCYP24-1 and TaCYP26-2, and testing the
chaperone or cooperative roles of all three ER isoforms, TaCYP23-1, TaCYP24-1,
TaCYP26-2, in wheat.
5.4.5 Chloroplast Cyps
Eight chloroplast Cyps in rice and five in wheat have been identified. The R55, F60,
and H126 were replaced by various residues and W121 by V/N/ I in different isoforms
(Tables 5.1; 5.3). The residues essential for CN binding, N- and O-glycosylation are not
conserved in a majority of these Cyps. Further studies are needed to test whether the
alterations influence PPIase activity. No insertion is present between α-helix-II and β-
strand-III in these Cyps. Insertions (11 or 24 aa) are present in several rice and wheat
Cyps between β-strand-IV to β-strand-V, located in the pocket (β-strands III to β-strand-
VI) for CsA binding and PPIase activity (Pflugl et al., 1993). The PPIase activity of
TLP40 with this insertion has been confirmed (Fulgosi et al., 1998), thus the relevant
wheat and rice isoforms may retain PPIase activity. The C/T targeting signals occurred
in all Cyps, with variable lengths. The leucine zipper motif involved in dimerisation
occurred in TaCYP46-2 and OsCYP47; their likely orthologue AtCYP38 with this motif
plays a critical role in the assembly and maintenance of PSII supercomplexes (Fu et al.,
2007; Sirpio et al., 2008). The orthologue of TaCYP26-3 and OsCYP27 - AtCYP20-2 -
plays an important role in catalysing the correct folding and integration of proteins in
the thylakoid membrane (Edvardsson et al., 2003; Romano et al., 2004b). The
orthologue of TaCYP26-1 and OsCYP25, AtCYP20-3 functions in repairing the
photodamaged PSII (Cai et al., 2008). Thus TaCYP26-1, TaCYP26-2 and TaCYP46-2
are excellent candidates for further study in this direction.
5.4.6 Nuclear Cyps
Four nuclear Cyps in rice and one in wheat have been identified. The R55 is replaced
by K, W121 is replaced by H, Y or R, and R69 is replaced by N or G or deleted in
different isoforms (Tables 5.1; 5.3; Fig 5.5D). N71 is replaced by D or S or T.
AtCYP59 containing T corresponding to R55, H corresponding to W121, deletion at
R69 for CN binding, and T corresponding to N71 has chaperone and/or PPIase activity
(Gullerova et al., 2006); thus the rice and wheat isoforms with such substitutions may
167
also be PPIase active or glycosylated. Eight amino acid insertion corresponding to the
plant Cyps insertion between α-helix-II and β-strand-III occurs in OsCYP49, OsCYP70,
and TaCYP70. A major insertion (10-32 aa) exists between β-strand-IV and β-strand-V
in OsCYP63 and OsCYP65, located in the pocket (β-strands III to β-strand-VI) for CsA
binding and PPIase activity (Pflugl et al., 1993). AtCYP59 with this insertion shows
chaperone and /or PPIase activity, so the insertion may not influence PPIase function.
The Arg/Lys rich domain for interaction with RNA (reviewed in He et al., 2004) and
involved in nuclear localisation and connecting transcription to pre-mRNA processing
(Gullerova et al., 2006) occurs in all these nuclear Cyps. The orthologue of TaCYP70
and OsCYP70, AtCYP63, has been confirmed to be localised in nucleus and play a role
in regulating the phosphorylation/dephosphorylation of signal recognition proteins and
other spliceosome components (Lorkovi et al., 2004). Further study should be
conducted on wheat and rice isoforms in this context.
5.4.7 Mitochondrial Cyps
Four mitochondrial Cyps (one in rice, three in wheat) have been identified. The
residues R55, F60, H126 are replaced by various residues in several isoforms, R69 by
F/L, and W121 by D in all isoforms (Tables 5.1; 5.3; Fig 5.5D), suggesting D may also
be involved in CsA contact. Further, N71, N108, T73 and S110 are replaced by D, I or
G in all Cyps. Four amino acid deletion occurs between α-helix-II and β-strand-III in
all, while some Cyps in other cellular locations present an insertion in this area (Saito et
al., 1999; Johnson and Bhave, 2004b).
5.4.8 Chromosomal locations of Cyps in rice and wheat
The twenty-seven rice loci encoding up to thirty-three Cyps are scattered on all
chromosomes except 12. The maximum number (six) is on Chromosome 6, and only
one on Chromosomes 5, 10, and 11. The distance between two Cyps on the same
chromosome arms ranges from 1014bp to 9.9Mb. The putative positions of two wheat
Cyps have been identified, TaCYP26-1 being on the short arm of 3A and TaCYP23
assessed to be on the long arms of chromosomes 1 and 7 groups but experimentally
placed on the long arm of chromosomes 7A, 7B and 7D (see section 4.3.3). The loci for
TaCYP23, identified by cDNA probes BE590822 on chromosome 1 and BE406148 on
chromosome 7, may be duplicates sharing high identity on cDNA level.
168
In summary, the Cyps in wheat and rice with substituted residues at PPIase activity
sites, CsA contact and glycosylation sites need to be tested for PPIase activity. The
functions of the likely equivalents of cytosolic TaCYP46-1, ER localised TaCYP24-1
and TaCYP26-2, chloroplast TaCYP26-1, TaCYP26-2, TaCYP46-2 and nuclear
TaCYP70 have been reported in other organisms but not in wheat or rice. This work is
essential due to diverse functions of such isoforms. Cyp with uncertain cellular location
may have roles in stress response, induced by drought. The ER-localised Cyps may co-
operate with each other to perform chaperone functions for regulating storage protein
deposition processes as well as numerous other proteins, as shown in literature.
Currently, only one ER isoform has been characterized (TaCYP23-1, as part of this
thesis), and there is no information on TaCYP24-1 and TaCYP26-2. Due to particular
interests in the ER-localised isoforms in storage protein processes, further study should
focus on the roles of all three such isoforms - TaCYP23-1, TaCYP24-1 and TaCYP26-2
- and their interactions with candidate proteins including seed storage proteins, using
systems such as the yeast-two hybrid. All identified TAs in wheat can be used for gene
mapping and expression analyses.
169
Chapter 6
A COMPARATIVE MOLECULAR AND PHYLOGENETIC ANALYSIS OF THE PROTEIN
DISULPHIDE ISOMERASE (PDI) FAMILY IN RICE AND WHEAT
170
6 A comparative molecular and phylogenetic analysis of the
protein disulphide isomerase (PDI) family in rice and wheat
6.1 Abstract
The protein disulphide isomerase (PDI) are a family of proteins containing thioredoxin
(TRX) domains that catalyse the formation and isomerisation of disulphide bonds
during protein folding processes and have additional chaperone and regulatory roles.
The ER-localized PDI isoform has been shown to have specific functions in segregation
of proglutelin and prolamin polypeptides in rice and to be differentially expressed in the
wheat endosperm. Other functions of PDI include development of ovule structure and
embryo sac and guidance of pollen tube in Arabidopsis, regulation of gene translation,
and protection from the pathogen (fungi) infection in wheat. The ER-localized PDI in
rice has been shown to have specific functions in segregation of proglutelin and
prolamin polypeptides. Our previous work showed that the PDI isoforms in wheat on
group 4 chromosomes are orthologous to the rice PDI gene close to the esp2
(endosperm storage protein) locus that regulates the seed storage protein body
formation. Using the reported loci encoding PDIs in Arabidopsis as queries to search
the rice genomic database and wheat TA database, 28 putative PDI members encoding
by 19 loci in rice (seven loci possibly encoding alternate splice forms) and 27 putative
PDIs in wheat were identified. They fell into five groups, i.e. PDIL1 (6, rice; 6, wheat);
PDIL2 (7, rice; 5, wheat); PDIL5 (6, rice; 13, wheat); QSOXL (2, rice; 1, wheat); APRL
(7, rice; 2, wheat). The gene structures in rice ranged from 3-exons to 15-exons; the
lengths of exons ranged from 22 bp to 1054 bp and those of introns from 74 bp to 1345
bp. The encoded proteins showed that in addition to the three reported PDIs, five more
putative ER-localised PDIs were identified in wheat. Expression patterns for three
reported PDI homologues (TaPDIL1-1, TaPDIL1-2, TaPDIL1-3), show firstly, that
there is no silencing of the genes during polyploidy in developing seeds, stems and
leaves, and are consistent with PDI playing a role in vegetative tissues as well as in the
developing endosperm.
171
6.2 Introduction
The protein disulphide isomerases (PDI) are a group of proteins containing thioredoxin
(TRX) domains that interact with nascent polypeptides to catalyse the formation and
isomerisation of disulphide bonds during protein folding processes (Creighton et al.,
1980). (see sections 1.5 and 1.6 for details). PDI was first found to catalyse both the
oxidation and isomerisation of disulfide bonds of nascent polypeptides in the ER
through the refolding of denatured glyceraldehyde-3-phosphate dehydrogenase (Cai et
al., 1994). PDI also functions in other cellular compartments, and participates in
regulation of actin nucleation and cell-to-cell interaction through activation of integrins
(reviewed in Noiva, 1999). Other members in PDI group have functions in apoptosis
during ER stress caused by accumulation of misfolded proteins (Jeong et al., 2008) and
peptide binding and protein retention in the ER (reviewed in Kim et al., 2009). The
larger family of PDI-like proteins, termed PDIL, is very diverse, with five major classes
including PDI, quiescin-sulfhydryl oxidase-like (QSOXL) and adenosine 5’-
phosphosulfate reductase-like (APRL) groups (Jacquot et al., 2002; Houston et al.,
2005).
The complexity of the PDIL family, consisting of 22 members in Arabidopsis, gives a
clue to the diversity of plant PDILs. Six PDI members are upregulated in response to
ER stress induced by treating seedlings with chemicals (Lu and Christopher, 2008a),
one member is required for normal seed development as a chaperone (Farquharson,
2008; Ondzighi et al., 2008) and one member has functions in embryo sac maturation
and pollen tube guidance (Wang et al., 2008). The chloroplast localised PDI in
Arabidopsis interacts with other proteins to regulate enzymes associated with starch
metabolism (Lu and Christopher, 2006; 2008b). The ER-localised PDI, associated with
the esp2 (endosperm storage protein) locus, has specific functions in segregation of
proglutelin and prolamin polypeptides in rice (Takemoto et al., 2002). Our previous
work showed that the three PDI homeoalleles in wheat are located on group 4
chromosomes, and their putative orthologue in rice is close to the esp2, hence they may
have similar functions.
This chapter describes the use of computational tools to identify the complete PDIL
family in rice (Oryza sativa ssp japonica cv. Nipponbare) from Rice Annotation
172
Release 6, and in wheat (Triticum aestivum L.) from the Plant Transcription Assembly
database; analysis of gene structures of PDILs in rice; phylogenetic relations of PDILs
in rice, wheat and Arabidopsis, identification of grain quality QTLs that co-localise at
the genes encoding PDILs in rice and wheat, and preliminary expression analysis of
genes encoding PDIs in wheat.
6.3 Results
6.3.1 Identification of genomic sequences of PDIL genes in rice
A search for PDIL family members in rice (Oryza sativa ssp japonica cv. Nipponbare)
was conducted in the Rice Genome Annotation Database Release 6
(http://rice.plantbiology.msu.edu/osa1.shtml) using the reported genomic sequences of
PDIL loci Arabidopsis (Houston et al., 2005) as queries (methods detailed in section
2.19). This led to identification of nineteen PDIL loci, resulting in 28 coding sequences
(CDS; exons contigues) due to some potential alternative splice forms (Table 6.1).
Eleven loci encoded single splice forms, seven potentially encoded two splice forms
(LOC_Os01g23740, LOC_Os02g34940, LOC_Os02g51850, LOC_Os03g17860,
LOC_Os05g06430, LOC_Os05g47930, LOC_Os07g34030) and LOC_Os09g27830
presented three splice forms (Fig. 6.1; alternative splice forms shown in Appendix IV).
In summary, of the possible alternative forms, (i) OsPDIL1-3 was shorter than
OsPDIL1-3a by 159 bp in exon I; (ii) OsPDIL2-1 was shorter than OsPDIL2-1a in
intron IX by 3 bp and longer in exon X by 3 bp; (iii) OsPDIL2-2 was shorter than
OsPDIL2-2a in exon IX by 4 bp and longer than it in intron IX-exon X by 466 bp; (iv)
OsPDIL5-1 and OsPDIL5-1a differed from exon III to the 3’-end; (v) OsPDIL5-4 was
longer than OsPDIL5-4a in the entire exon I-intron IV section by 2405 bp; (vi)
OsQSOXL1 was longer than OsQSOXL1a in partial intron XI-exon XII section by 131
bp; (vii) OsAPRL3 was longer than OsAPRL3a in part of intron III to exon IV by 598
bp; and (viii) OsPDIL2-3, OsPDIL2-3a and OsPDIL2-3b varied from exon VI to the 3’-
end.
173
6.3.2 Identification of expressed sequences of PDIL genes in rice
Twenty-two expressed PDILs were also retrieved from the rice Transcript Assembly
(TA) database (see section 2.19). Of these, 13 were the same as those reported by
Houston et al. (2005) from an EST database, six reported by Houston et al. (2005) were
replaced by other names, and three were new. Of the 22, 18 were comprised of single
ESTs and four comprised of multiple ESTs. Six of the 28 CDSs (exon contigs)
mentioned above did not have a corresponding TA. Of these, five corresponded to
OsPDIL2-2a, OsPDIL2-3b, OsPDIL5-1a, OsPDIL5-4a and OsQSOXL1a, suggesting
these splice forms may not be expressed, or at least not in the tissues that the ESTs were
from. No EST corresponding to OsPDIL5-3 was noted, suggesting a similar situation.
6.3.3 Predictions of intron/exons structures of rice PDIL genes
The lengths of PDIL genes ranged from 2171 bp (encoded at LOC_Os08g31814) to
6757 bp (encoded at LOC_Os07g34030), while the CDSs ranged from 378 bp
(OsPDIL5-1a) to 1692 bp (OsPDIL1-4) (Table 6.1). The gene structures generated by
comparisons of genomic sequences with CDS varied from 3-exons-2-introns (OsPDIL5-
1a) to the fairly complex 15-exons-14-introns (OsPDIL5-4) (Fig 6.2); these are
discussed separately later.
6.3.4 Grouping of putative PDIL proteins in rice
The putative protein sequences translated from the 28 CDSs ranged from 13.9 to 62.2
kDa (125 to 563 amino acids) (Table 6.1). Based on characteristics of the PDI active
site(s) (see section 1.5), these were divided into five groups, i.e., PDIL1, PDIL2, PDIL5,
QSOXL (quiescin-sulfhydryl oxidase-like) and APRL (adenosine 5’-phosphosulfate
reductases-like), as defined by Houston et al. (2005) (Fig. 6.2, Fig. 6.4). The lowest
inter-group identity was 6% (OsPDIL5-1a to OsPDIL1-3) and the highest was 37 %
(PDIL1-1 to PDIL5-3) (Table 6.2). The putative amino acid sequences including signal
peptides (SP) and functional domains were compared to those in wheat and other plants;
(detailed in section 6.3.7). The key characteristics of each group are described below.
174
Fig 6.1 Rice PDIL gene structures The gene structures were generated by comparison of genomic sequences with corresponding CDSs downloaded from the Rice Annotation Release 6 (http://rice.plantbiology.msu.edu/osa1.shtml) with Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn). The blue lines indicate extra region in genomic sequences compared to the CDS, green boxes indicate exons and grey lines indicate introns. Regions marked in red indicate sections encoding the putative catalytic site(s).
175
Table 6.1 PDIL gene family in rice
Name Locus GS
a
(bp) CDS
b
(bp) Exons cDNA/ ESTs
c
Protein (aa/KDa)
cellular locations
d
OsPDIL1-1e Os11g09280 3817 1539 11 AK068268 512/56.9 CP
1, ER
2
OsPDIL1-2 Os04g35600 3072 1554 11 AY739308 517/57.3 n/a1, ER
2
OsPDIL1-3 Os02g34940 4751 1479 10 AK073161 492/53.3 CP1, ER
2
OsPDIL1-3a Os02g34940 1638 10 AK072252 545/59.1 CP1, ER
2
OsPDIL1-4 Os02g01010 4864 1692 12 AK071514 563/62.2 CP1, ER
2
OsPDIL1-5 Os06g06790 4643 1602 12 AK073970 533/59.0 CP1, ER
2
OsPDIL2-1 Os05g06430 3360 1101 11 AK098931 366/39.9 CP1
,ER2
OsPDIL2-1a Os05g06430 1104 11 AK099341 367/40.0 CP1 ,ER
2
OsPDIL2-2 Os01g23740 3882 1116 11 AK066111 371/40.7 CP1
,ER2
OsPDIL2-2a Os01g23740 951 9 n/a 316/34.4 CP1
,ER2
OsPDIL2-3 Os09g27830 4984 1326 9 AK062254 442/47.3 CP1,ER
2
OsPDIL2-3a Os09g27830 1188 7 AK072941 395/43.5 CP1,ER
2
OsPDIL2-3b Os09g27830 1083 7 n/a 360/38.9 CP1,ER
2
OsPDIL5-1 Os03g17860 2351 444 4 AK063663 147/16.3 CP1,ER
2
OsPDIl5-1a Os03g17860 378 3 n/a 125/13.9 CP1,ER
2
OsPDIL5-2 Os04g35290 3012 1275 5 AK069367 424/47.0 PM1,ER
2
OsPDIL5-3 Os02g34530 2311 1281 5 n/a 426/47.1 EC1, ER
2
OsPDIL5-4 Os07g34030 6757 1458 15 AK099660 485/54.4 C1,n/a
2
OsPDIL5-4a Os07g34030 1191 12 n/a 396/45.0 C1,n/a
2
OsQSOXL1 Os05g47930 5020 1542 12 AK121660 513/57.8 ER1,2
OsQSOXL1a Os05g47930 1557 11 n/a 518/58.4 ER1,2
OsAPRL1 Os07g32570 3296 1428 3 XM478340
(AU062587) 475/50.6 CP1,2
OsAPRL2 Os06g11740 2126 849 4 AY739306 282/31.6 n/a1.2
OsAPRL3 Os02g51850 2545 936 4 XM_467860 (CA763645) 311/34.8 CP
1, ER
2
OsAPRL3a Os02g51850 624 3 CB644946 207/22.7 CP1, ER
2
OsAPRL4 Os08g31814 2171 846 4 AY739307 281/30.7 CP1,n/a
2
OsAPRL5 Os03g59170 3123 906 4 AK073308 301/33.0 CP, ER2
OsAPRL6 Os12g35640 3146 903 4 CA767512 300/33.0 n/a1,2
aGS: length of genomic sequence;
bCDS: length of coding sequence or exon contigs;
cTAs
identified in this work and also reported as ESTs (Houston et al., 2005) (not shaded) or TAs identified only in this work (shaded);
dCL (cellular locations) predicated using
1WoLF PSORT
Prediction (http://psort.ims.u-tokyo.ac.jp) and 2TargetP 1.1
(http://www.cbs.dtu.dk/services/TargetP) (C: cytosol; CP: chloroplast; EC: extracellular; PM: plasma membrane);
ereported PDI (Takemoto et al., 2002; Johnson et al., 2006). Loci shown in
bold possibly encode >1 splice type. All TAs/ESTs in column 6 were used as queries for generating the maps of PDIL chromosomal locations (Fig. 6.6).
The OsPDIL1 group comprised of six members (OsPDILl-1, OSPDIL1-2, OsPDIL1-3,
OsPDIL1-3a, OsPDI1-4, OsPDI1-5), encoded by five loci. The lengths/molecular
weights of the putative full-length proteins (including any SPs) ranged from 492 aa/53.3
kDa (OsPDIL1-3) to 563 aa/62.2 kDa (OsPDIL1-4) and their identities (excluding any
alternative splice forms) ranged from 21% to 62% (Tables 6.1, 6.2). The SPs were
predicted by SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP) and all of
176
them contained the SPs as reported human PDI (hPDI, P07237) (Ferrari and Soling,
1999) (Fig 6.4). The cleavage site was predicted to be between residue A25 and E26
(OsPDIL1-1), G23 and V24 (OsPDIL1-2), A61 and A62 (OsPDIL1-3), A22 and E23
(OsPDIL1-3a), A22 and S23 (OsPDIL1-4), and A22 and R23 (OsPDIL1-5). Based on
this, the mature proteins were determined to be 52 to 59 kDa. The PDIL1 genes
encoded proteins with two active sites, one each at the N- and C-termini, connected by
334-341 aa (Fig 6.2). OsPDIL1-1 represents the rice PDI gene reported to have an
important role in seed storage protein body (PB) formation (Takemoto et al., 2002)
(section 1.7). The product of OsPDIL1-3 was longer than OsPDIL1-3a by 53 aa at the
N-terminus (discussed later). These two may be alternative splice forms, and TAs
(AK073161 and AK072252) were noted encoding these (Table 6.1), indicating both are
expressed. Two putative active sites with the conserved motif WCGHC were conserved
in four members (OsPDIL1-1, OSPDIL1-2, OsPDIL1-3, OsPDIL1-3a) (Fig 6.2).
OsPDIL1-4 contained an N-terminal ‘WCAHC’ and a C-terminal ‘WCGHC’, while
OsPDIL1-5 contained N-terminal ‘WCERS’ and C-terminal ‘WCVDC’. The C-
terminal KDEL of all these members has also been reported in Arabidopsis (Houston et
al., 2005). Using TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP) and WoLF
PSORT (http://psort.ims.u-tokyo.ac.jp) (section 2.22), all six members were predicted to
be in the ER lumen. However, five out of six were also predicted to be in the
chloroplast (Table 6.1), at >90% with program TargetP 1.1 or >50% degree of
confidence with program WoLF PSORT.
The OsPDIL2 group had seven members, each with two active sites (each WCGHC) at
the N-terminus, connected by 114-131aa (Fig 6.2). The putative proteins ranged from
316 aa/ 34.4 kDa (OsPDIL2-2a) to 442 aa/47.3 kDa (OsPDIL2-3) with identities
(excluding alternative forms) of 28 to 82% (Tables 6.1, 6.2). All exhibited SPs, with
potential cleavage between A29 and D30 (OsPDIL2-1, OsPDIL2-1a), A27 and A28
(OsPDI2-2, OsPDIL2-2a), A18 and S19 (OsPDIL2-3, OsPDIL2-3a, OsPDIL2-3b) (Fig
6.4), the estimated sizes of mature proteins being 31 to 46 kDa. OsPDIL2-1 and
OsPDIL2-1a were represented by TAs AK098931 and AK099341, respectively,
indicating both are expressed, and their putative proteins differed by two residues, i.e.,
an insertion of N (at position 311 of OsPDI2-1a) and a K-to-R substitution (at position
312 of OsPDIL2-1a). OsPDIL2-2 was longer than OsPDIL2-2a by 56 aa at the C-
terminus. OsPDIL2-3, OsPDIL2-3a and OsPDIL2-3b may be different splice forms, the
177
first two being represented by an EST/TA. OsPDIL2-3a and OsPDIL2-3b were shorter
than OsPDIL2-3 by 47 aa and 82 aa at he C-terminus, respectively. The C-terminal
NDEL (OsPDIL2-3), LLST (OsPDIL2-3a), CLLR (OsPDIL2-3b), TFSS (OsPDIL2-1,
OsPDIL2-1a, OsPDIL2-2), SAAK (OsPDIL2-2a) are novel, compared to VASS
(AtPDIL2-1), KDDL (AtPDIL2-2), or KDEL (AtPDIL2-3) in Arabidopsis (Houston et
al., 2005). All six may be in the ER lumen or chloroplast.
The six OsPDIL5 members ranged from 125 aa/13.9 kDa to 485 aa/54.4 kDa with
identities (excluding alternative forms) of 7-74% (Table 6.1, Table 6.2). Each had an
N-terminal active site, WCGHC (OsPDIL5-3 and OsPDIL5-4) or a variant, e.g.,
WCKHC (OsPDIL5-1 and OsPDIL5-2), WCYWS (OsPDIL5-4 and OsPDIL5-4a) (Fig
6.2, Fig. 6.4). Alignment with an hPDI member (Erp18, O95881) showed all members
contained the SP (Jeong et al., 2008) with cleavage sites predicted between A29 and
E30 (OsPDIL5-1 and OsPDIL5-1a), A35 and E36 (OsPDIL5-2) and A28 and G29
(OsPDIL5-3). Four members (OsPDIL5-1, OsPDIL5-1a, OsPDIL5-2, OsPDIL5-3)
contained predicted cleavage sites, leading to mature proteins of 12 kDa (OsPDIL5-1a)
to 44 kDa (OsPDIL5-3). OsPDIL5-1 and OsPDIL5-1a differed in length and sequence
from position 121 to C-terminus, while OsPDIL5-4 and OsPDIL5-4a differed from the
N-terminus to residue 98. The C-terminal GKNI of OsPDIL5-4 and OsPDIL5-4a are
reported in Arabidopsis (Houston et al., 2005), while the LQDS, NLRL, AHEE and
AHED (of OsPDIL5-1, OsPDIL5-1a, OsPDIL5-2, OsPDIL5-3, respectively) are new.
The proteins are predicated to be located in ER, and/or chloroplast, and other locations.
The OsQSOXLs included possible alternative splice forms OsQSOXL1 and
OsQSOXL1a, encoding 513 aa/57.8 kDa or 518 aa/58.4 kDa proteins with 98% identity
(Table 6.1; Table 6.2; Fig 6.2). They contained an SP (Thorpe et al., 2002) with the
predicted cleavage site between A30 and R31 and mature proteins of 55 kDa
(OsQSOXL) or 56kDa (OsQSOXL1a). The C-terminal KNWN and LKKI of
OsQSOXL and OsQSOXLa are novel, compared to those of AtQSOX1 (REKE) and
AtQSOX2 (PRRR) (Houston et al., 2005). The proteins are predicted to be located in
the ER.
The seven members with variant active sites at the C-terminus (WCPFC; APRL1) or N-
terminus (WCPFS; 6 others) were grouped in the OsAPRL group. They ranged from
207 aa/ 22.7 kDa to 475 aa/50.6 kDa, with identities (excluding alternative forms) of
178
13-66% (Table 6.1, Table 6.2; Fig. 6.2). The chloroplast transit peptide predicted by
ChloroP 1.1 Server (http://www.cbs.dtu.dk/services/ChloroP) was noted in OsAPRL1
(63 aa), as in Arabidopsis (Gutierrez-Marcos et al., 1996), leading to a mature protein of
44 kDa. The C-terminal NSLR (OsAPRL1), PSTS (OsAPRL2), NELR (OsAPRL3),
LVFS (OsAPRL3a), PSLS (OsAPRL4), SRQA (OsAPRL5) and AVLD (OsAPRL6)
were novel, compared to those of APRLs (AtAPR1 and AtAPR3, NLVR; AtAPR2,
NLLR; AtAPR4, SSSQ; AtAPR5, SDQS; AtAPR6, SASQ; AtAPR7, SQSA) in
Arabidopsis (Houston et al., 2005). OsAPRL3 and OsAPRL3a may be alternative
forms, with a TA (CB644946) noted for OsAPRL3a. APRL1 was predicted to be only
in chloroplast and others predicted in chloroplast or ER.
OsQSOXL1
OsQSOXLa
OsPDIL2-3b
KDEL341aa
OsPDIL1-1341aa KDEL
OsPDIL1-3a337aa KDEL
OsPDIL1-4334aa KDEL
OsPDIL1-5
114aa TFSS
NDEL131aaOsPDIL2-3
131aa LLST OsPDIL2-3a
CLLR 131aa
OsPDIL1-3
340aa KDEL
OsPDIL1-2
KDEL341aa
OsPDIL2-1
OsPDIL2-1a OsPDIL2-2
SAAKOsPDIL2-2a
114aa
LQDSOsPDIL5-1
NLRLOsPDIL5-1a
AHEE OsPDIL5-2
AHED OsPDIL5-3
GKNIOsPDIL5-4
GKNIOsPDIL5-4a
KNWN
LKKI
NSLR OsAPRL1PSTS
OsAPRL2NELR
OsAPRL3
LVFS OsAPRL3aPSLS
OsAPRL4SRQA
AVLDOsAPRL5
OsAPRL6
Fig 6.2 Schematic of the structures of PDILs in rice. Colour bars indicate positions of the catalytic site(s), i.e., WCGHC (blue); WCAHC (red); WCERS (yellow); WCVDC (orange); WCKHC (pink); WCYWS (green); WCPAC (purple); WCPFC in OsAPRL1 or WCPFS in other OsAPRLs (dark blue). The number above the line indicates the length between two active sites. The C-terminal tetrapeptide sequences are indicated.
179
Table 6.2 Identities (%) between putative amino acid sequences and between the coding sequences of PDILs in rice*
Id (%) CDS PDIL1 PDIL2 PDIL5 QSOXL APRL
AA 1-1
1-2
1-3
1-3a
1-4
1-5
2-1
2-1a
2-2
2-2a
2-3
2-3a
2-3b
5-1
5-1a
5-2
5-3
5-4
5-4a 1 1a 1 2 3 3a 4 5 6
PDIL1
1-1 62 56 56 37 35 26 26 26 23 29 27 25 12 11 36 37 22 21 28 28 25 16 17 12 15 17 15
1-2 53 61 61 38 33 25 25 25 23 29 27 25 12 10 35 35 22 22 26 26 25 15 16 11 15 17 15
1-3 56 61 90 21 22 18 18 18 16 23 21 19 8 6 22 22 19 19 24 23 25 16 17 12 15 17 16
1-3a 51 55 90 40 37 22 22 23 21 27 25 23 10 9 34 34 23 20 24 24 26 16 17 12 14 17 15
1-4 25 23 24 22 46 23 23 22 19 25 23 21 10 8 27 28 22 19 27 27 23 14 16 11 14 14 15
1-5 15 15 16 15 22 21 21 21 18 25 23 21 10 8 27 29 23 20 27 27 25 14 16 11 14 16 15
PDIL2
2-1 14 12 13 12 11 9 99 82 71 31 36 37 20 17 29 27 19 20 21 20 22 20 23 17 20 20 21
2-1a 14 12 13 12 11 9 99 82 71 31 36 37 20 17 29 27 19 20 21 20 22 20 23 17 20 20 21
2-2 14 11 13 12 11 8 82 81 85 32 37 38 21 18 28 28 20 20 21 21 22 20 23 18 20 20 20
2-2a 13 11 12 11 11 7 71 70 85 28 33 34 25 21 25 25 17 16 19 18 19 23 26 21 23 23 24
2-3 15 13 14 12 12 11 19 19 20 19 78 78 14 12 29 29 20 20 26 25 21 15 16 13 16 15 15
2-3a 15 12 13 12 12 11 20 20 21 21 68 87 15 13 30 29 22 22 23 22 22 19 21 14 18 19 19
2-3b 14 12 13 11 11 11 21 21 22 23 77 75 17 14 28 28 21 21 22 22 19 18 20 15 20 18 18
PDIL5
5-1 7 6 5 5 5 4 14 14 15 18 8 9 10 84 13 13 10 9 10 10 8 13 13 19 16 13 12
5-1a 6 6 5 4 5 4 12 12 13 15 7 8 9 83 12 12 8 7 8 8 7 11 11 16 14 11 11
5-2 19 18 20 18 14 12 14 14 15 14 13 15 13 9 9 74 24 24 24 24 27 17 18 14 17 19 19
5-3 19 19 19 17 14 13 14 14 13 13 13 15 13 8 8 68 25 24 24 24 29 18 19 13 17 19 19
5-4 4 5 5 4 4 4 4 4 3 2 5 5 4 1 1 6 6 82 22 21 21 16 18 13 16 19 18
5-4a 5 6 5 5 5 5 4 4 4 3 6 5 5 2 2 7 7 80 22 21 20 17 20 13 15 20 19
QSOXL
1 9 9 8 8 8 6 9 9 9 9 10 10 10 5 5 8 8 9 12 98 25 22 23 15 21 22 20
1a 9 8 8 8 8 6 9 9 9 9 10 10 10 5 5 8 8 9 12 98 24 22 23 15 21 22 20
APRL
1 7 7 7 6 8 9 6 6 5 5 7 8 6 2 2 8 8 3 5 7 7 19 19 13 17 19 20
2 8 7 8 7 6 6 10 10 10 11 6 6 7 10 8 9 8 2 4 8 8 8 66 44 42 40 38
3 8 6 8 7 7 8 10 10 10 12 7 7 9 9 7 10 8 2 3 8 8 7 55 58 45 40 39
3a 7 4 5 5 6 7 8 8 8 9 5 6 6 13 10 8 7 1 2 5 5 4 37 56 41 31 31
4 6 6 6 6 5 7 9 9 9 10 7 7 8 8 6 7 7 1 3 6 6 6 36 32 30 32 31
5 6 6 7 6 7 9 8 8 9 10 7 7 9 7 6 9 9 2 4 6 6 6 27 28 20 21 51
6 7 6 6 5 6 8 8 8 9 10 7 8 8 8 7 11 9 2 4 6 6 5 21 24 18 19 39
*Identities (%) of CDS shown above the diagonal and identities of putative protein sequences shown under the diagonal. The identities were obtained based on alignments of CDSs (data not shown) obtained from the Rice Genome Annotation Database Release 6 (http://rice.plantbiology.msu.edu/osa1.shtml) and the predicted amino acids sequences (Fig. 6.2) by pairwise comparisons in BioEdit. Lines between columns are not aligned between row 1 and the rest of the table.
180
6.3.5 Analysis of the rice PDIL gene structures
As explained in section 6.3.3, the PDIL genes had a range of intron/exon structures.
The lengths of individual exons varied from 22 to 1054bp (Table 6.3) and those of
introns from 74 to 1345 bp (data not shown). All putative intron/exon junctions were
the archetypical GT/AG except for intron IV of OsQSOXL1 and OsQSOXL1a
(AG/GC; Appendix V). Genes for OsPDIL1-1 and OsPDIL1-2 had the same 10-exon
structure, the sizes of exons II, III, IV, VIII, IX were conserved and the two active sites
(WCGHC) were encoded in exon II and IX, respectively (Fig 6.1). OsPDIL1-3 and
OsPDIL1-3a presented 9 exons and sites WCGHC in exon II and XIII. Exon II (325
bp) of OsPDIL1-3 (or OsPDIL1-3a) seemed to be split in OsPDIL1-1 and OsPDIL1-2
(exon II and III, 31 and 288 bp) (Fig 6.2). Sizes of exon III to VIII of OsPDIL1-3 or
OsPDI1-3a were extremely similar to those of exon IV to IX of OsPDIL1-1 or
OsPDIL1-2 (e.g. exon III of OsPDIL1-3 or OsPDIL1-3a, and exon IV of OsPDIL1-1 or
OsPDIL1-2, are 189 bp). The identities of CDS and putative amino acid sequences of
OsPDIL1-3 to OsPDIl1-3a were both 90%. Further, the CDS (i.e. contigue of exons I-
IX) of OsPDIL1-3 showed highest identities to CDS (exon I-X) of OsPDIL1-1 (56%)
and OsPDIL1-2 (61%), with respective protein identities of 56 or 61% (Table 6.2),
suggesting LOC_Os11g09280, LOC_Os04g35600 and LOC_Os02g34940 may be
duplicates dispersed on three chromosomes. OsPDIL1-4 and OsPDIL1-5 had 12 exons
and conserved lengths of exons II, V and VI but varied in lengths/positions of most
introns.
The genes encoding OsPDIL2-1, OsPDIL2-1a, OsPDIL2-2 and OsPDIL2-2a showed
conservation of sizes of exons II to VIII (Table 6.3; Fig 6.2). The identities (on CDS
and protein levels both) between the pairs were 99% and between OsPDIL2-1 and
OsPDIL2-2 were 82% (Table 6.2). LOC_Os05g06430 and LOC_Os01g23740 thus
appear to be duplicates. The gene encoding OsPDIL2-3 has a different structure and
more limited identity to OsPDIL2-1 and OsPDIL2-2 (19-20% on CDS, 31-32% on
protein level).
The 5-exon genes encoding OsPDIL5-2 and OsPDIL5-3 showed conservation in length
(but not sequence) of exon IV (308bp) and 68% identity on CDS and 74% on amino
acid sequence level (Fig 6.1; Table 6.3). LOC_Os04g35290 and LOC_Os02g34530
181
encoding these are also possibly duplicate genes. In comparison, the OsPDIL5-1
encoding gene has 4 exons and limited identity to these (8-9% on CDS, 13% on amino
acid levels).
Genes encoding QSOXL1 (or 1-a) has 11 (or 12) exon structures and limited identity to
all other groups. Gene encoding OsAPRL1 had only 3 exons, while five others had four
exons. Genes those encoding OsAPRL2, OsAPRL3, OsAPRL3a and OsAPRL4
showed conserved length (but not sequence) of exon II (188 bp); and OsAPRL2 and
OsAPRL3 were also conserved in length of exon III (125 bp) (Table 6.3). The identity
between OsAPRL3 and OsAPRL3a was 56% on CDS and 58% on amino acid sequence
level, very similar to that between OsAPRL2 and OsAPRL3 (55% on CDS, 66% on
amino acid level). In comparison, the identities of OsAPRL2 and OsAPRL3 encoding
genes with OsAPRL1, OsAPRL4, OsAPRL5 and OsAPRL6 were much lower (7-36%)
at CDS levels. Thus the loci LOC_Os06g11740 and LOC_Os02g51850 may be
duplicate genes (Table 6.2).
182
Table 6.3 Summary of exon sizes (bp) of PDIL genes in rice*
Exon
I Exon
II Exon
III Exon
IV Exon
V Exon
VI Exon
VII Exon VIII
Exon IX
Exon X
Exon XI
Exon XII
Exon XIII
Exon XIV
Exon XV
OsPDIL1-1 203 31 288 189 126 138 120 118 113 213
OsPDIL1-2 179 31 288 189 126 138 123 118 113 249
OsPDIL1-3 158 325 189 126 138 117 118 113 195
OsPDIL1-3a 317 325 189 126 138 117 119 113 195
OsPDIL1-4 503 130 169 121 81 87 75 67 81 135 57 186
OsPDIL1-5 488 130 166 119 81 87 72 69 78 141 54 117
OsPDIL2-1 173 85 98 56 118 28 92 157 125 100 69
OsPDIL2-1a 173 85 98 56 118 28 92 157 125 103 69
OsPDIL2-2 188 85 98 56 118 28 92 157 125 100 69
OsPDIL2-2a 188 85 98 56 118 28 92 157 129
OsPDIL2-3 126 159 114 182 100 177 136 157 175
OsPDIL2-3a 126 159 114 182 100 402 105
OsPDIL2-3b 126 159 114 182 100 177 225
OsPDIL5-1 126 59 176 83
OsPDIl5-1a 126 59 193
OsPDIL5-2 240 104 371 308 252
OsPDIL5-3 252 104 374 308 240
OsPDIL5-4 41 91 95 58 185 58 41 75 57 67 147 225 162 78 78
OsPDIL5-4a 23 183 55 41 75 57 67 147 225 162 78 78
OsQSOXL1 224 25 93 180 75 69 74 154 41 147 438 22
OsQSOXL1a 224 25 93 180 75 69 74 154 41 147 475
OsAPRL1 58 316 1054
OsAPRL2 162 188 125 374
OsAPRL3 177 188 125 446
OsAPRL3a 177 188 259
OsAPRL4 252 188 125 281
OsAPRL5 183 182 125 416
OsAPRL6 192 176 125 410
The sizes of exons （from start codon to stop codon） were calculated based on the gene structures (Fig 5.1) generated with gene structure display server
program (http://gsds.cbi.pku.edu.cn).
183
6.3.6 PDIL coding sequences in wheat
The TIGR Plant Transcript Assemblies database (http://plantta.jcvi.org) was queried as
in Section 6.3.1 and led to identification of twenty-seven TAs encoding putative PDILs
in wheat. These ranged from 584-2444 bp and encoded putative proteins of 14.7 kDa
(130 aa) to 63.7 kDa (585 aa) (including SPs), grouping into the same five groups as
rice, based on TRX catalytic site number, position, residues and other motifs. These
comprised of TaPDIL1 (6 members), PDIL2 (5), a larger PDIL5 (13), QSOXL (1) and
APRL (2) (Fig 6.3; Table 6.4). Their key characteristics are described below. The
putative amino acid sequences were compared to those in rice and other plants (see
later; section 6.3.7).
The putative full-length TaPDIL1 proteins ranged from 492 aa/53.3 kDa (PDIL1-5),
512-516aa/56kDa (PDIL1-1, 1-2, 1-3, 1-4) to 585 aa/63.7 kDa (PDIL1-6). The SPs
were predicted by SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP) with
cleavage sites between A25 and E26 for four members (TaPDIL1-1, TaPDIL 1-2,
TaPDIL 1-3, TaPDIL 1-4), A22 and E23 (TaPDIL1-5) or A28 and A29 (TaPDIL1-6).
Based on this, the mature proteins were estimated to be 52 to 61 kDa. All PDIL1s had
two conserved TRX sites WCGHC, one each at the N- and C-terminus, connected by
336-341 aa, and a conserved C-terminal KDEL. TaPDIL1-1, TaPDIL1-2 and TaPDIL1-
3 were 100% identical to the cDNAs (wPDI1, wPDI2, wPDI3) reported previously by
M. Bhave’s group corresponding to the homeologous genes on group 4 chromosomes,
(Johnson et al., 2001) or CPDI-4A, CPDI-4B, and CPDI-4D (Ciaffi et al., 2006).
TaPDIL1-4 showed 83-84% on coding sequence level and 85% on amino acid level
with TaPDIL1-1, TaPDIL1-2 and TaPDIL1-3, indicating it is the duplicate of the three
homeologous genes.
The five identified TaPDIL2 members encoded putative proteins of 210 aa/22.5 kDa to
440 aa/47.2 kDa (Fig 6.3, Table 6.4). Their predicted SPs had cleavage sites between
G30 and D31 (TaPDIL2-1, TaPDIL2-2), A29 and D30 (TaPDIL2-3), or A22 and L23
(TaPDIL2-4, TaPDIL2-5), the mature proteins being 20-49 kDa. The proteins had two
conserved N-terminal active sites (WCGHC), connected by 114 aa (TaPDIL2-1,
TaPDIL2-2, TaPDIL2-3) or 132 aa (TaPDIL2-4, TaPDIL2-5). The proteins were
predicted to be in the ER; however, the C-terminal AFSS (TaPDIL2-1), KSGN
(TaPDIL2-2), TFSS (TaPDIL2-3), AART (TaPDIL2-4) or NDEL (TaPDIL2-5) are
184
new, compared to PDIL2s in Arabidopsis (Houston et al., 2005). The pairs TaPDIL2-2
and TaPDIL2-1 (98.5% identity) and TaPDIL2-4 and TaPDIL2-5 (95.7% identity) may
be duplicates or homeoalleles of each other.
The 13 TaPDIL5s (Fig 6.3, Table 6.4) encoded putative proteins ranging from 130
aa/14.7 kDa to 485 aa/54.4 kDa, with most predicted to be in ER. All were also
predicted to have a SP except for TaPDIL5-13, with the cleavage sites predicted
between residues A32 and E33 (TaPDIL5-1, TaPDIL5-3), A30 and E31 (TaPDIL5-2),
A39 and E30 (TaPDIL5-4), G30 and D31 (TaPDIL5-5), A25 and E26 (TaPDIL5-6,
TaPDIL5-7), A30 and E31 (TaPDIL5-8), A24 and E25 (TaPDIL5-9), A22 and L23
(TaPDIL5-10), G24 and G25 (TaPDIL5-11), A35 and E36 (TaPDIL5-12). The mature
proteins were thus estimated to be 12 to 44 kDa. The conserved site WCGHC’ occurred
in nine members, with three (TaPDIL5-1, TaPDIL5-2, TaPDIL5-3) having WCKHC
and one (TaPDIL5-13) WCYWS.
Only one QsOXL member was identified, as in rice, the putative 301 aa/32.9 kDa
protein exhibiting an active site WCPAC and being predicted in ER and elsewhere. The
SP was predicted as above, its cleavage site between A25 and L26 and the mature
protein being 31 kDa. SPs have been reported in Arabidopsis and human QsOXLs
(Thorpe et al., 2002). The C-terminal LAWK is new, compared to QsOXL in
Arabidopsis (Houston et al., 2005).
Two APRL members were identified, with active site WCPYC at C-terminus
(TaAPRL1) (compared to WCPFC in rice) or WCPFS at N-terminus (TaAPRL2;
identical to rice). The predicted proteins were 463 aa/50.5 kDa (TaAPRL1) or 282
aa/31.6 kDa (TaAPRL2), the former predicted in chloroplast only and the latter in
diverse locations. Chloroplast transit peptides predicted by ChloroP 1.1 Server
(http://www.cbs.dtu.dk/services/ChloroP) showed TaAPRL1 contained this peptide (64
aa), as in Arabidopsis (Gutierrez-Marcos et al., 1996), the mature proteins being
estimated at 45 kDa. The C-terminal SSSQ and PSTS of TaAPRL1 and TaARPL2,
respectively, are new, compared to those of Arabidopsis APRLs (Houston et al., 2005).
185
TaPDIL5-12
TaPDIL5-11
KDEL339aaTaPDIL1-1
TaPDIL1-2
TaPDIL1-3
340aa KDEL
341aa KDELTaPDIL1-5
336aa KDELTaPDIL1-6
114aa AFSS
KSGN114aaTaPDIL2-2
114aa TFSS TaPDIL2-3
AART 132aaTaPDIL2-4
TaPDIL2-5
TaPDIL1-4
TaPDIL2-1
132aa NDEL
ENLNTaPDIL5-1
DVDRTaPDIL5-2
EDEL
TaPDIL5-3LQDS
TaPDIL5-4MRGV
TaPDIL5-5HWTG
TaPDIL5-6PPRG
TaPDIL5-7VHDE
TaPDIL5-8LEDL
TaPDIL5-9EIGS
TaPDIL5-10AHQE
AHEE
GKNITaPDIL5-13
LAWKTaQsOXL1
NSLRTaAPRL1
PSTSTaAPRL2
Fig 6.3 Schematic of the structures of PDILs in wheat. Colour bar indicates the five residues in the catalytic sites: i.e. WCGHC (blue); WCKHC (pink); WCYWS (green); WCPAC (purple); WCPYC (red); WCPFS (dark blue). The number above line indicates the length between two active sites. The C-terminal tetrapeptide sequences are indicated.
186
Table 6.4 PDIL family in wheat
Name TA number TA
(bp) Exons Protein
(aa/KDa) Cellular location
a
TaPDIL1-1b TA63630_4565 2395 10 515/56.6 CP
1, ER
2
TaPDIL1-2b TA63634_4565 1884 10 512/56.4 CP
1, ER
2
TaPDIL1-3b TA63632_4565 1893 10 515/56.6 CP
1, ER
2
TaPDIL1-4 AK068268 1878 n/a 512/56.8 CP1, ER
2
TaPDIL1-5 AK073161 1816 n/a 492/53.3 ER1,2
TaPDIL1-6 TA76289_4565 1972 n/a 585/63.7 ER1,2
TaPDIL2-1 TA55349_4565 1430 n/a 367/40.3 CP1, ER
2
TaPDIL2-2 CK218055 1130 n/a 259/28.4 CP1, ER
2
TaPDIL2-3 NM_185280 1101 n/a 366/39.9 CP1, ER
2
TaPDIL2-4 TA67032_4565 713 n/a 210/22.5 ER1,2
TaPDIL2-5 TA67031_4565 1577 n/a 440/47.2 CP1, ER
2
TaPDIL5-1 CA627363 600 n/a 148/16.7 M1,2
TaPDIL5-2 TA74480_4565 745 n/a 149/16.6 V1, ER
2
TaPDIL5-3 CA626641 584 n/a 130/14.7 n/a1, M
2
TaPDIL5-4 AK063663 686 n/a 147/16.3 n/a1, ER
2
TaPDIL5-5 TA55340_4565 825 n/a 202/22.3 CP1, ER
2
TaPDIL5-6 DR736972 1143 n/a 288/31.7 CP1, ER
2
TaPDIL5-7 CV775131 724 n/a 212/23.2 CP1, ER
2
TaPDIL5-8 TA71882_4565 1719 n/a 417/46.6 EC1, ER
2
TaPDIL5-9 DR739734 1144 n/a 234/25.9 EC1, ER
2
TaPDIL5-10 CK204275 856 n/a 202/21.8 EC1, ER
2
TaPDIL5-11 TA76778_4565 1570 n/a 414/46.1 PM1, ER
1,2
TaPDIL5-12 AK069367 1645 n/a 423/47 PM1, ER
1,2
TaPDIL5-13 AK099660 1947 n/a 485/54.4 n/a1,2
TaQSOXL CK207082 1068 n/a 301/32.9 ER1,2
TaAPRL1 TA69080_4565 2444 n/a 463/50.5 CP1,2
TaAPRL2 AY739306 1134 n/a 282/31.6 n/a1,2
aCellular locations predicated using
1WoLF PSORT Prediction (http://psort.ims.u-tokyo.ac.jp)
and 2TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP). (CP: chloroplast; M: mitochondria;
EC: extracellular; PM: plasma membrane; V: vacuole). bTaPDI1-1, TaPDIL1-2, TaPDIL1-3
reported as wPDI1, wPDI2, and wPDI3 (Johnson et al., 2001) or CPDI-4A, CPDI-4B, and CPDI-4D ( Ciaffi et al., 2006), respectively.
6.3.7 Comparison of the putative PDIL amino acid sequences of rice and wheat to
each other and to other plant PDILs
The putative amino acid sequences of PDIL1s, translated from the CDSs in rice and
TAs in wheat, were aligned with human PDI (hPDI; P07237) for predicting secondary
structures (Fig 6.4A). All PDIL1s in rice and wheat contained SPs at N-terminus for
ER targeting (Price et al., 1991), four domains (a, b, b’, and a’) (Freedman et al., 2002;
Wilkinson and Gilbert, 2004), the x-linker between b’ and a’, and the c-extension with
an ER-localisation signal (Freedman et al., 2002) (section 1.4.3). The length of x-linker
appears conserved, with only a two-residue insertion in OsPDIL1-2. The SPs varied in
length and sequence, from 22 aa (OsPDIL1-2) to 61 aa (TaPDIL1-3). Based on
187
alignment with hPDI1, all domains showed small (1-5 residue) insertions/deletions in
various rice and wheat PDIL1 members, some shared between >1 proteins, others
unique to a particular protein. The size of x-linker was conserved with only a two-
residue insertion at position 498-499 in OsPDIL1-2. The active sites CGHC in domain
a were variant in two out of six OsPDIL1s, as mentioned above, but conserved in all
TaPDIL1s, and the active site in a’ was conserved in all except OsPDIL1-5 (WCVDC).
Variants have also been noted in AtPDIL1-3 (WCGAC) and AtPDIL1-5 and AtPDIL1-
6 (WCARS) (Houston et al., 2005). The c extension ranged from 26-47 aa and the C-
terminal ER-localization KDEL were conserved in PDIL1 members in both plants, as
also in Arabidopsis.
The sequences of putative PDIL2 proteins were aligned with human PDI hP5 (Hayano
and Kikuchi, 1995) (Genbank Q15084) and alfalfa P5 (Ferrari and Soling, 1999) (Fig
6.4B) to predict secondary structures. Four out of seven members in rice (OsPDIL2-1,
OsPDIL2-1a, OsPDIL2-2, OsPDIL2-2a) and four out of five members in wheat
(TaPDIL2-1, TaPDIL2-2, TaPDIL2-3, TaPDIL2-4) showed similar secondary structures
as for alfalfa P5, with three domains (ao, a, D) (Ferrari and Soling, 1999). OsPDIL2-3
and TaPDIL2-5 showed similar secondary structures as reported for human P5, with
four domains (ao, a, b, c) (Ferrari and Soling, 1999); while OsPDIL2-3a was short of c,
and OsPDIL2-3b was short of b and c. OsPDIL2-3 and TaPDIL2-5 contained C-
terminal NDEL for ER retention (He et al., 2004).
The 19 putative PDIL5 members were aligned with human ERp18 (Liu et al., 2003)
which appears to correspond to this group (Fig 6.4C). OsPDIL5-1 and OsPDIL5-1a
contained only the a domain (Jeong et al., 2008; Kim et al., 2009), while others
(OsPDIL5-2, OsPDIL5-3, OsPDIL5-4, OsPDIL5-4a) contained a domain and extension
at C-terminus. The section of a domain was highly similar, with two main insertions
(>6 residues) in OsPDIL5-4, OsPDIL5-4a and TaPDIL5-13. The active sites were
variant in both rice and wheat, i.e., WCKHC (OsPDIL5-1, OsPDIL5-2, TaPDIL5-1,
TaPDIL5-2, TaPDIL5-3, TaPDIL5-4) or WCYWS (OsPDIL5-4, OsPDIL5-4a,
TaPDIL5-13) found in AtPDIL5-1 or AtPDIL5-3 and AtPDIL5-4 (Houston et al.,
2005). OsPDIL5-4a and TaPDIL5-13 contained a 90 aa extension at N-terminus.
Compared to
188
ERp18, 12 members contained extra sequences at C-terminus, as noted in AtPDIL5-2,
AtPDIL5-3 and AtPDIL5-4.
The three putative QSOXL proteins aligned with human HsQSOXL (HSU97276)
showed they had a/a’ domain (WAV-KVA) of PDI with the motif WCPAC, and an
ERV1 domain (DFI-CYL) including its three main parts: (i) section with the active site
CXDC; (ii) section with the consensus motif (HNXVNXR/KL) for interacting with the
ADP motif of FAD group; (iii) section with consensus motif (ELCXXC) (Thorpe et al.,
2002) (Fig 6.4). TaQSOXL was highly homologous to rice with active site WCPAC
found in AtQSOX1 and AtQSOX2 in Arabidopsis (Houston et al., 2005). AtQSOXl do
not have a typical ER retention signal (Fig 6.4D) but have been isolated from ER.
There is also evidence for gene duplications in this family before duplication of the a/a’
TRX domains, and continued evolution through alternative splicing, e.g., in human
HsQSOX1 (Thorpe et al., 2002).
The sequences of the nine putative APRL proteins were aligned with those of
Arabidopsis (PRH-19, PRH-26, PRH-43, Gutierrez-Marcos et al., 1996; or AtAPR1,
AtARP3, AtAPR2, respectively, Houston et al., 2005) in order to find active sites and
degrees of homology (Fig 6.4E). OsAPRL1 and TaAPRL1 presented the active sites
WCPFC or WCPYC at C-terminus and lack the N-terminal site, as in Arabidopsis
APRLs (AtAPR1, AtARP3, AtAPR2) (Gutierrez-Marcos et al., 1996; Houston et al.,
2005), while others contained WCPFS at the N-terminus but not at C-terminus, as in
case of other Arabidopsis APRLs (AtAPRL4, AtAPRL5, AtAPRL6, AtAPRL7)
(Houston et al., 2005).
189
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hPDI -----------------------------------------------------MLRR---ALLCLAVAALVR-----------------------------------ADAPEEE------------------------------------
AtPDIL1-1 -----------------------------------------------------.AM.----GFT.FSILVLS---------------------------------LC.SSIRS.E-----------------------------------
AtPDIL1-2 -----------------------------------------------------.AFK----GFACFSIL.LLS--------------------------------LFVSSIRS.------------------------------------
AtPDIL1-3 -----------------------------------------------------.ASSSTSIS.L.F.SFILLLVNSRAENASSGSDLD-EELAFLAAEESK----EQSHGGGSYHEEEHDHQHRDFENYDDLEQGGGEF-HHGDHGYEEE
AtPDIL1-4 -----------------------------------------------------.AF.--VL..FSLT.L.IFSAVSPSFAASSSDDVDDEDLSFLEDLKEDDVPGADSLSSSTGFDEFEGGEEEDPDMYNDDDDEEGDFSDLGNPDSDPL
AtPDIL1-5 ---------------------------------------------MSLIPKPISKVSTFTFI.LILLSFTIIIAYSSPDSNVESNEP--------------GFDSDLDQLLAVDEQLQEDRPEQQ----------------------SEA
AtPDIL1-6 ---------------------------------------------MLTKPKPNSKFS-ILFTFL.LLSF.IFVARSS-DVAVEAGSE--------------EELDDLEQLLAVDEQLQEERPEQQ----------------------SEA
OsPDIL1-1 -----------------------------------------------------.AISKAWIS.L..L.VVLSAPAAR-----------------------------AEE.AAA.------------------------------------
OsPDIL1-2 -----------------------------------------------------.AVN---LV.SF.L.I.IS------------------------------------SS.TAVG----------------------------------V
OsPDIL1-3 MWPRAPATPPPPPWPSKPSAASRSALRRLDLDDGRRQGTEGEENHAPLLCSPA.ASS---TAFAA.F.L.LL-----------------------------------.SSAAA.G-----------------------------------
OsPDIL1-3a -----------------------------------------------------.ASS---TAFAA.F.L.LL-----------------------------------.SSAAA.G-----------------------------------
OsPDIL1-4 -------------------------------------------------------M.SRSL..VALATL.LHASASAS----------DDDLDYLIDNA--------D.I.ANDPDGWLQEGSPD------DDDDDDLFHHG------QA
OsPDIL1-5 ----------------------------------------------------MRA..VVA.AAV.LLF.V.AVARLDLDDDG-------------------DDSEVLDELLAVDE--EEERGELGGG-----------------GEAAAA
TaPDIL1-1 -----------------------------------------------------.AISKVWIS.L..L.VVLSAPAAR-----------------------------AEE.AAA.------------------------------------
TaPDIL1-2 -----------------------------------------------------.AICKVWIS.L..L.VVLSAPAAR-----------------------------AEE.AAA.------------------------------------
TaPDIL1-3 -----------------------------------------------------.AICKAWIS.L..L.VVLSAPAAR-----------------------------AEE.AAAA------------------------------------
TaPDIL1-4 -----------------------------------------------------.AISKAWIS.L..L.VVLSAPAAR-----------------------------AEE.AAA.------------------------------------
TaPDIL1-5 -----------------------------------------------------.ASS---TAFAA.F.L.LL-----------------------------------.SSAAA.G-----------------------------------
TaPDIL1-6 --------------------------------------------------MAA.PMPRSLL.IL.LATP.LILPLAAAAVPTSNP---DIDLEYLIKNAGL-----DDPT.ATTATDPEDDGAPDFPGLDADYDDEDLFGDDDGPEEDSS
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hPDI -------DHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESD--LAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLRDG--AAAESLVESSEVAVIGFFK
AtPDIL1-1 ---TETKEF..T.DHT..TDTINK.DFIV............Q.....E...SA.SSNVPPVV...I..S..TNREF.T..E.Q.F....I....GK.-VQ..NGP...EG..TY...QS...SAEIKS--ADD.SEV.SDKK.V.V.I.P
AtPDIL1-2 ----ETKEF..T.DH...T.TISK.DFIV...........QK.....E...SE.SSHNPPLA...I..S..ANKEF.NE.KIQ.F..L.IL...GKS-VQD.NGP...EG..TY...QS...SVEIKS--ADS.TEV.GEKN.VAV.V.P
AtPDIL1-3 PLPPVDEKD.A..T.D..T.FVGNNSFAM.........A.Q..T....A..TE..G---LAA...I.....G.--...K.EIQ.F..VFL.VD.EMR--.T.EGE.TK.G..T....KAS.SIHNITT--KEE..RVLSAEPKL.F..LN
AtPDIL1-4 PTPEIDEKD.V.IKER..TDVIENNQ.V............QS......A..TE..ED--GVV...I.....NE--...E.R.Q.F..LL..VD.EH---.P..G..TKET..T.V..KI..GVYN.TT--LDD..KVLT.GNKV.L.YLN
AtPDIL1-5 ETVSKAQRI..E.NGDYTKRVIDGNEFVM.LG.....ARSAE.M.RF.E..TA..EI..SVLM..I.G--DRYSKI.SELEIK.F..LLL.V..---TSLT.NG.SS.E...I.VQ.K..APII..NT--VDE.PRFLDKYHTF.L.L.E
AtPDIL1-6 ETVSKAQRI.VE.NGD.TKRLIDGNE.VM.LG.....ARSAE.M.RF.E..TD..EI..SVLM..I.G--.RYSKV.S.LEIK.F..LLL.V..---TSQS..G.FSSEE..I.VQ.K..ASTIK.DT--VDE.SGFLKKHHTFIL.L.E
OsPDIL1-1 EGGDAAAEA..T.DADG.D..V.K.PFMV............K.....E...QE.SKHDPP.V......ND.KNKP..TK.EIQ.F..L.I...QGKN-IQ..KGP...EG..EY...QV...SKEIKS--PED.TN.IDDKKIYIV.I.S
OsPDIL1-2 DATEELKEA..T.DAG..S.VV.K.PFIV.K..........Q.....E...SI.RKNELPVV......YN.RNKE.KDK...YS.....IMK..GSD-VRG.GGP....G..EY..RQV...SLK.ES--AEE.AHS.VDKG.ILV.V.P
OsPDIL1-3 -------EA..T.DAG..T.VVG..DFIV...........NQ.....EA..AA.RSHDPPVV......SADLNRG..GEH..Q.....RIL.DRGAR-SHN.AGP.D.AG..AY..RQA...SVEIAASASPP.ADSIANDG.V.V.V.P
OsPDIL1-3a -------EA..T.DAG..T.VVG..DFIV...........NQ.....EA..AA.RSHDPPVV......SADLNRG..GEH..Q.....RIL.DRGAR-SHN.AGP.D.AG..AY..RQA...SVEIAASASPP.ADSIANDG.V.V.V.P
OsPDIL1-4 QDHPIDET..FL.SAA..SDF..S.RHVM........A..Q....D..A..AD.SPLAHQVA........DT.--...K.D.Q.F...L..ID.-VP--.D.NGA.TKEA..S.VN.KLA.GVQNITT--VDE..KILTGEDK.ILAVLD
OsPDIL1-5 EAVRRAQSM....DND.ARR.VEENAEV.LLG.....ERSAQ.M.RF.E..AA.R.M..AVAF..L.G--.RYPKA.SAV..K.F..VLL.V..---TEHQF.GLHTK.A..T.VR.K..AP.SRIQS--KDS..EFLKKDQTFAV.L..
TaPDIL1-1 EAA-AAPEA..T.HAD..DD.I.K.PFI.............S.....E...QL.SKHDPA.V......ND.KNKP..GK.E.Q.F..L.I....GKN-IQ..KGP...EG..EY...QV...SKEIKA--PED.T-YL.DGKIHIV.V.T
TaPDIL1-2 EAA-AAPEA..T.HAD..DD.I.K.PFI.............S.....E...QL.SKHDPA.V......ND.KNKP..GK.E.Q.F..L.I....GKN-IQ..KGP...EG..EY...QV...SKEIKA--PED.T-YL.DGKIHIV.V.T
TaPDIL1-3 EEA-AAPEA..T.HAD..DD.I.K.PFI.............S.....E...QL.SKHDPA.V......ND.KNKP..GK.E.Q.F..L.I....GKN-IQ..KGP...EG..EY...QV...SKEIKA--PED.T-YL.DGKIHIV.V.T
TaPDIL1-4 EGGDAAAEA..T.DADG.D..V.K.PFMV............K.....E...QE.SKHDPP.V......ND.KNKP..TK.EIQ.F..L.I...QGKN-IQ..KGP...EG..EY...QV...SKEIKS--PED.TN.IDDKKIYIV.I.S
TaPDIL1-5 -------EA..T.DAG..T.VVG..DFIV...........NQ.....EA..AA.RSHDPPVV......SADLNRG..GEH..Q.....RIL.DRGAR-SHN.AGP.D.AG..AY..RQA...SVEIAASASPP.ADSIANDG.V.V.V.P
TaPDIL1-6 HPSAADEA...L.TAA..TSV...RRHVM...........R....H..A..SA.AEQ.VDVA........DH.--...AH..Q....LL..ID.-VP--RD.AGE.TK.A..A.IS.KL...VQN.TT--ADE..KI.TGDD...LAYLD
A
a
TRX
b
190
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hPDI DVESDSAKQFLQAAEAIDDIPFGIT----SNSDVFSKYQLDKDG--VVLFKK-FDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGG----EIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDS-DHTDNQR
AtPDIL1-1 KLSGSEFDS.MAI..KL-RSELDFAHT--.DAKLLPRGESSVT.PV.R...P-...QFVDSK-DFDG.A.EK.V.ESSI..ITV.DKDPNNHPYVIKFFESTN.KAM..INFTGEGAESLK.KYREV.T.N..QG.SFLLG--.AENS.G
AtPDIL1-2 KLSG.EFDS.MAL..KL-RADYDFAHT--LDAKFLPRGES-VE.PA.R...P-...LFVDSK-DFNG.A.EK.V.ESSI...TV.DSDPNNHPYVAKFFESPA.KAMM.VNFTGATAEALK.KYREV.T.N.DQS.AFLVG--.AESS.G
AtPDIL1-3 SLVG-.ESEE.A..SRL-EDDLSFYQTASPDIAKLFEIETQVKRPAL..L..E-E.KLAR.D.NF..TAIAE.VSA.KV....N..REG.S-----LIFESSVKNQ.ILFA.ANE-SEKH.PTLREV.K.....FV.VYVQMDNEDYGEA
AtPDIL1-4 SLVG-VEHDQ.N..SKA-EDDVNFYQTVNPDVAKMFHLDPESKRPAL..V..E-E.KISH.D..FV.SA.VS.VSA.K.A..SV..RE...-----EIFESAIKKQ..LFVTKNE-SEKV.TE.QE..K.....LI.VSV.LDNEDYGKP
AtPDIL1-5 KF.GSE-HNEFVK.AKS-.DEIQFIETRD.DVAKLLFPD.KSNNVFIG.V.PE-A.RYTVYD.SYKM.KI.E.LGS.KF..FTKL..TNTV-----WVYSSPVKLQVMLFS.ADD-FQKLAQPLEDI.RK..S.LM..YV.ITNENLAMP
AtPDIL1-6 KS.DS.GHDEFVK.ASL-.NEIQFVETSSIDVAKLLFPN.KTNNVF.G.V.TE-A.KYTSYD.PCQA.KIVE.LNS.KF...TKL..SNTV-----RVYSSPVKLQVMVFS.TDD-FESLAQPLEDI.RK..S.LML.Y..ISNENLAMP
OsPDIL1-1 ELSGTEYTN.IEV..KL-RSDYDFGHT--LHANHLPRGDAAVERPL.R...P-...LVVDSK-DFDVTA.EK..DASST.K.VT.DKNPDNHPYLLKFFQSSAAKAM...NF.TGPFESFK.VYYG...E..D.EIKFL.G--.IEAS.G
OsPDIL1-2 EFAGMEYEN.MVV..KM-RADYDFFHT--.DASILPRGDQSVK.PI.R...P-...LFVDS.-DFG.DA.EK..EVSGF.M.VTYDADPTNHK.LERYYSTPSSKAM..VSFGDDRIESFK.QIHE..RK.S.NNISFL.G--.VA.AD.
OsPDIL1-3 ELSGSEFES.MAV..KM-RADYDFRHT--TDAG.LPRGDRTVR.PL.R...P-...LFVDSQ-DFDRDA.EK..ESSGF.T.VT.DTSP.NQKYLLKYFDNAG.KAM...SF.DDRAEEFRTQ.HE..NQYSANNISFL.G--.V.AS.G
OsPDIL1-3a ELSGSEFES.MAV..KM-RADYDFRHT--TDAG.LPRGDRTVR.PL.R...P-...LFVDSQ-DFDRDA.EK..ESSGF.T.VT.DTSP.NQKYLLKYFDNAG.KAM...SF.DDRAEEFRTQ.HE..NQYSANNISFL.G--.V.AS.G
OsPDIL1-4 SLSG-AHSDEIA..SRL-EDAINFYQTSNPDVAKLFHLDPAAKRPSL..L..QEE.KLTFYD.PFKASAIA..VSA.K....NTL.QE...-----SIFDNPIKKQI.LFVVANE-SSKF.PI..E.SK.....L..V.VERDNEEVGEP
OsPDIL1-5 NF.GAE-YEEFVK.ATS-ENEVQFVETNDR.VAKILFPGIASEEQFLG.V.SE-P.KFEK.N.AFEEKEIIQ.VEL.KF..ITV..DLNSG-----KVYGSPIKLQVFTFAEAYD-FEDLE.MIQEV.RG..T..ML.YV.TAEEKLAKP
TaPDIL1-1 EFSGTEFTN..EL..KL-RSDYDFGHT--VHANHLPRGDAAVERPL.R...P-...LVVDSK-DFDVSA.EK..DASST.K.VT.DKNPDNHPYLLKYFQSNAPKAM...NF.TGPFESFK.AYYG.V.E.S..DVKFL.G--.IEAS.G
TaPDIL1-2 EFSGTEFTN..EV..KL-RSDYDFGHT--VHANHLPRGDAAVERPL.R...P-...LVVDSK-DFDVSA.EK..EASST.K.VT.DKNPDNHPYLLKFFQSNAPKAM...NF.TGPFESFKKAYYG.V.E.S..DVKFL.G--.IEAS.G
TaPDIL1-3 EFSGTEFTN..EV..KL-RSDYDFGHT--VHANHLPRGDAAVERPL.R...P-...LVVDSK-DFDVSA.EK..DASST.K.VT.DKNPDNHPYLLKFFQTNAPKAM...NF.TGPFESFK.AYYG.V.E.S..DVKFL.G--.IEAS.G
TaPDIL1-4 ELSGTEYTN.IEV..KL-RSDYDFGHT--LHANHLPRGDAAVERPL.R...P-...LVVDSK-DFDVTA.EK..DASST.K.VT.DKNPDNHPYLLKFFQSSAAKAM...NF.TGPFESFK.VYYG...E..D.EIKFL.G--.IEAS.G
TaPDIL1-5 ELSGSEFES.MAV..KM-RADYDFRHT--TDAG.LPRGDRTVR.PL.R...P-...LFVDSQ-DFDRDA.EK..ESSGF.T.VT.DTSP.NQKYLLKYFDNAG.KAM...SF.DDRAEEFRTQ.HE..NQYSANNISFL.G--.V.AS.G
TaPDIL1-6 HLSG-AHSDE.A..SRL-EDTISFYQTTSPDVAKLFHIDPEAKRPS...L..E-E.KLTV.D..FRASAIAE.VSA.KI..ITTL.QE...-----AIFDNPIKKQI.LFAVAKE-SPQF.PII.ET.K.....L..V.VERDNEEVGEP
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hPDI ILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFL--EGKIKPHLMSQERAGDWDKQPVKVPVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDH--ENIVIAKMDSTANEVE--AVKVHS-FPTL
AtPDIL1-1 AFQY...EESQV.LIIIQ----TADDK.YLKTNVEVDQ.ESWVKD.K--D...A..KK..PIP-AENNE....V.SDSLD.IVLNSG...LL..........QK....L.EVAVS.QSD--SSV....L.A...DFPKDTFD.KG-...I
AtPDIL1-2 AFQY...EESQV.LIIIQ----TPDNK.YLKVNVEVDQ.ESWFKD.Q--D..VAV.KK..PIP-AENNE....V.AESLD.IV.KSG...LI..........QK....L.EVALSFQND--PSVI...L.A...DIPSDTFD.KG-...I
AtPDIL1-3 VSG...VTGAAPKVLVYTGN--.DMRKFILDG...VNN.KTLAED..--AD.L..FYK.DPLP-ENNDGD...I..N..DEIVL..S.D.LL.I........QSFE..YN...KYL.GI--DSL.V....G.S..HP--RA.ADG-...I
AtPDIL1-4 VA.Y..VSGNGPKLIGYTGN--.DP.KYFFDG.IQSDK.KI.GED..--ND.L..FYK.DPIP-EKNDED..IV..D..DEIVL.DS.D.LL.V........QA.E.MYN..AKHLRSI--DSL..T...G.T..HP--KA.AEG-...I
AtPDIL1-5 F.IL..IEAGNKTV.AAFDN--NLNSKYLLESDPSPNS.E...SGLA--H.TVSRYYR.EPVP-.NENASIVTV...T.DGLVLNSRE..LL.VHT...VN.EA.SKQIE..AKHF.--GF..L.F.RI.AS...HT--KLQ.DDKY.II
AtPDIL1-6 F.TL..IEDAKKTV.AAFDN--NLNSKYLLESDPSPSN.E...FGLA--H.TVSAYYK..PIP-.NQNAS.VAV..RT.DE.VLRSSE..LL.VHT...IN.EA.SKQVE..SQHF.--GF..L.F.RI.AS...HP--KLT.DD-Y..I
OsPDIL1-1 AFQY...REDQV.LIIIQ----DGESK.FLKAHVEPDQ.VSWLKEYF--D..LS.FRK.EPIP-EVNDE....V.AD.VH.FV.KSG...L............K....L.EAAT.L.SD--KDV......A...D.P-SEFD.QG-Y...
OsPDIL1-2 VFQY...RESDV.LLFV.----AS.GK-YLNPTMDPDQ.IPWLKQYIVEY.NLT.YVK.EPIP-KVND.....V.AD.ID.IV.NSG...LL..........RKF.L.LEEIAVSLQ.D--QD.......G.V.DIP-TDFT.EG-Y..I
OsPDIL1-3 AFQY....ES.V.L.FIL----ASKSK-YIKPTVEPDQ.LPYLKE.T--..TLA..VK.EPIP-EVND....TV.AD.LRE.V.NSG...LL..........QK....LEEVAVSL..D--.DV......G...D.P-SDFA.EG-Y.SM
OsPDIL1-3a AFQY....ES.V.L.FIL----ASKSK-YIKPTVEPDQ.LPYLKE.T--..TLA..VK.EPIP-EVND....TV.AD.LRE.V.NSG...LL..........QK....LEEVAVSL..D--.DV......G...D.P-SDFA.EG-Y.SM
OsPDIL1-4 VANY..ITGQ.TTVLAYTGN--.DARNFFLDG.ISV.N.KR.AED..--.E.LT.FYK.EPVP-ESNEGD..IV....LDQIVL..S.DALL.I........QE.E.TYN...KHLRGI--DSL......G....HP--RA.PDG-...I
OsPDIL1-5 F.TLY..EP-.K.T.TAFDT--SKGTKYLMEA.IN.KNLQD..LSL.--..TLP.YFR.EPVP-E-E.G.IEKV..RT.DSSVLESPQ...L.VH....VD.EAISKNVE..AKHFN.LGQT.LKF.RI.ASV..HP--KLQINN-Y...
TaPDIL1-1 AFQY....EDQA.LILIQ----DSDSK.FLK.QVE.GQ.VAWLKDYF--D..LT.FRK.EPIP-EANNE....V.AD.IH..V.KSG...LI...........K....L.EAAA.LQSE--.DV....I.A...D.P-GEFD.QG-Y...
TaPDIL1-2 AFQY....EDQA.LILIQ----DSDSK.FLK.QVE.GQ.VAWLKDYF--D..LT.FRK.EPIP-EANNE....V.AD.VH..V.KSG...LI...........K....L.EAAA.LQSE--.DV......A...D.P-SEFD.QG-Y...
TaPDIL1-3 AFQY....EDQA.LILIQ----DSDSK.FLK.QVE.GQ.VAWLKDYF--D..LT.FRK.EPIP-EANNE....V.AD.VH..V.KSG...LI...........K....L.EAAA.LQSE--.DV......A...D.P-SEFD.QG-Y...
TaPDIL1-4 AFQY...REDQV.LIIIQ----DGESK.FLKAHVEPDQ.VSWLKEYF--D..LS.FRK.EPIP-EVNDE....V.AD.VH.FV.KSG...L............K....L.EAAT.L.SD--KDV......A...D.P-SEFD.QG-Y...
TaPDIL1-5 AFQY....ES.V.L.FIL----ASKSK-YIKPTVEPDQ.LPYLKE.T--..TLA..VK.EPIP-EVND....TV.AD.LRE.V.NSG...LL..........QK....LEEVAVSL..D--.DV......G...D.P-SDFA.EG-Y.SM
TaPDIL1-6 VANY..IAGQ.TTVLAYTGN--.DA.KFFF.G.ISLDT.K..AQD..--.D.LT.SYK.DPVP-ESNDED...V...SLDQIVL..S.D.LL.V........QS.E..YN..AKYLRGI--DSL......G.N..HP--RA.PDG-...I
b’
x a’
TRX
191
610 620 630 640 650 660 670 680
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
hPDI KFFPASADR-TVIDYNGERTLDGFKKFLE-------SGGQDGAGDDDDLEDLEEAEEPDMEED-------DDQKAVKDEL
AtPDIL1-1 Y.KS..GN---.VV.E.D..KED.IS.VD---------KNKD-----------TVG..KK.--------EETTEE.....
AtPDIL1-2 Y.RS..GN---.VV.E.D..KED.IN.V.---------KNSEKKPTSHG.ESTKS...KKT--------EET--.A....
AtPDIL1-3 L...GGNKSFDP.AVDVD..VVELY...KKHASIPFKLEKPATPE--------PVISTMKSDE------KIEGDSS....
AtPDIL1-4 L....GNKTSEP.TVDTD..VVA.Y...RKHATIPFKLEKPASTESPKTAESTPKV.TTETKESPDSTTKSS.SDS....
AtPDIL1-5 LLYKSGEKE-KPLKLSTKLSAKDIAV.IN---------EELLKPKNGS---------------------------A....
AtPDIL1-6 LLYKTGEKE-NPLKLSTKSSAKDMAVLIN---------KEL-KWK.QS---------------------------G....
OsPDIL1-1 Y.VTP.GK---MVP.ESG..A.EIVD.IK---------KNKE---TAGQ-----.K.KAES---------APAEPL....
OsPDIL1-2 Y.YSS.GN---LLS.D.A..AEEIIS.IN---------ENR.PKAGAAAAVD.KTQIDAV..EVT-----SSSEP.....
OsPDIL1-3 Y.YSSGGN---LLP.D.-..AEEIID.IT---------KNK.SRPGEAT----------------------TTES.....
OsPDIL1-3a Y.YSSGGN---LLP.D.-..AEEIID.IT---------KNK.SRPGEAT----------------------TTES.....
OsPDIL1-4 L.Y..GKKSFEP.TFE.D..VVEMY..IKKHASIPFKLKRPDSSATKTEK.QST.STNLRG.R-------SSGTNF....
OsPDIL1-5 LLY..QDKS-NP.KLSKKSN.KDMA..VK---------EKLQIA.VETVA---------------------AGDI.....
TaPDIL1-1 Y.VTP.GK---KVS.E.G..A.EIVDYIK---------KNKE---TAGQAAAAAT.KAAEP---------AATEPL....
TaPDIL1-2 Y.VTP.GK---KVS.E.G..A.EIVDYIK---------KNKE---TAGQ---AAT.KAAEP---------AATEPL....
TaPDIL1-3 Y.VTP.GK---KVS.E.G..A.EIVDYIK---------KNKE---TAGQAAAADT.KAAEP---------AATEPL....
TaPDIL1-4 Y.VTP.GK---MVP.ESG..A.EIVD.IK---------KNKE---TAGQ-----.K.KAES---------APAEPL....
TaPDIL1-5 Y.YSSGGN---LLP.D.-..AEEIID.IT---------KNK.SRPGEAT----------------------TTES.....
TaPDIL1-6 L.Y..GKKSFEP.TFE.D..VVEMY...KKHAAIPFKLKRPDSSAAQTDDSSST..----G.K-------SSSSKP....
c
192
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
P5(Alfalfa MKMEMHQIWSRIALASFAFAILFVS------------VSADDVVVLTEENFEKEVGH-DKGALVEFYAPWCGHCKKLAPEYEKLPNSFKKAKSVLIAKVDCDEHKSVCSKYGVSGYPTIQWFPKGSLEPKKFEGPRTAESLAEFVNTEGG
AtPDIL2-1 --.AKS...FGF..L----.L.L..------------AV........DDS......K-..........................GA.................Q....T....................Q.Y....N..A...Y..K...
OsPDIL2-1 --.ATP..S-.K..---.SLL.L.AAAAAVS-----TA.....LA...ST......Q-.RA.......................GA.............................................Y..Q....A...Y..S.AA
OsPDIL2-1a --.ATP..S-.K..---.SLL.L.AAAAAVS-----TA.....LA...ST......Q-.RA.......................GA.............................................Y..Q....A...Y..S.AA
OsPDIL2-2 --.AIPR.SP.KT.PL..ALA.ALAWAFAAP----AFADG....A...ST......Q-.R........................GA........F....................................Y..Q.S..A..........
OsPDIL2-2a --.AIPR.SP.KT.PL..ALA.ALAWAFAAP----AFADG....A...ST......Q-.R........................GA........F....................................Y..Q.S..A..........
TaPDIL2-1 --.ATP..Y-.KT.---LPVL.LLAAAALYP----AAADG.E.LA...ST......Q-.R........................AA.............................................Y..Q....A.T.Y..S.AA
TaPDIL2-2 --.ATP..Y-.KT.---LPVL.LLAAAALYP----AAADG.E.LA...ST......Q-.R......................R.AA...........RG................................Y..Q....A.T.Y..S.AA
TaPDIL2-3 --.ATP..S-.K..---.SLL.L.AAAAAVS-----TA.....LA...ST......Q-.RA.......................GA.............................................Y..Q....A...Y..S.AA
TaPDIL2-4 ---MRPA.L--------.AIL.LLAAAAS-PAAAL-YSAGSP.LQ.NPN..K-K.LNANGVV....F........Q.T.IW..AAGVL.GVAT.AALDA.AHKELA--QQ..IR.F...KV.LP.KP-.VDY..A.DVKPIVN.ALSQVK
P5(Human) ----------------M.LLV.GLVSCTFFLAVNGLYS.S...IE..PS..NR..IQS.SLW............QR.T..WK.AATAL.DVVK.GAVDA.KHHSLG--GQ...Q.F...KI.GSNKNR.EDYQ.G..G.AIVDAALSALR
AtPDIL2-2 ---MYKS----------TVFPICCLLFALFDRGNALYG.SSP.LQ..PS..KSK.LNSNGVV....F.......QS.T.TW..VASTL.GIAT.AAIDA.AHKSV.--QD...R.F...KV.VP.KP-.IDYQ.A.D.K.ISQ.AIKQIK
AtPDIL2-3 ---MYKSPL--------TLLT.LTICFGFFDLSSALYG.SSP..Q..AS..KSK.LNSNGVV....F........A.T.TW..VA.IL.GVAT.AAIDA.AHQSAA--QD..IK.F...KV.VP.KA-.IDYQ.A.D.K.I.N.AYKQIK
OsPDIL2-3 ---MRPAVA--------.ALL.VAAAVAASPVSAL-YSAGSP.LQFNPN..KSK.LNSNGVV....F.......QQ.T.IW..AAGVL.GVAT.AALDA.AHKELA--QE..IR.F...KV.VP.KP-.VDYQ.A.DVKPIV..ALSQVK
OsPDIL2-3a ---MRPAVA--------.ALL.VAAAVAASPVSAL-YSAGSP.LQFNPN..KSK.LNSNGVV....F.......QQ.T.IW..AAGVL.GVAT.AALDA.AHKELA--QE..IR.F...KV.VP.KP-.VDYQ.A.DVKPIV..ALSQVK
OsPDIL2-3b ---MRPAVA--------.ALL.VAAAVAASPVSAL-YSAGSP.LQFNPN..KSK.LNSNGVV....F.......QQ.T.IW..AAGVL.GVAT.AALDA.AHKELA--QE..IR.F...KV.VP.KP-.VDYQ.A.DVKPIV..ALSQVK
TaPDIL2-5 ---MRPA.L--------.ALL.LLAAAAS-PAAAL-YSAGSP.LQ.NPN..K-K.LNANGVV....F........Q.T.IW..AAGVL.GVAT.AALDA.AHKELA--QQ..IR.F...KV.LP.KP-.VDY..A.DVKPIVN.ALSQVK
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
P5(Alfalfa ---------------------TNVKIATAPSHVVVLTPETFNEVVLDGTKDVLVEFYAPWCGHCKSLAPIYEKVAAVFKSEDD--VVIANLDADKYRDLAEKYDVSGFPTLKFFPKGNKAGEDYGGGRDLDDFVAFINEKSGTSRDAKGQ
AtPDIL2-1 ---------------------....L.AV.QN......DN.D.I...QN....................T.....T...Q.EG--.........AHKA.G...G............D....H..D.........S...........S...
OsPDIL2-1 ---------------------......AV..S.........DS....E.................H.......L.S.Y.QDEG--..........HTA.....G....................D...E.....K.....C.....S...
OsPDIL2-1a ---------------------......AV..S.........DS....E.................H.......L.S.Y.QDEG--..........HTA.....G....................D...E.....K.....C.....S...
OsPDIL2-2 ---------------------....L..I..S....G.DN.DSI...EN..I.............H.......L.S.Y.LD.G--..........HK......G...Y................D...E.....K.....C.....T...
OsPDIL2-2a ---------------------....L..I..S....G.DN.DSI...EN..I.............H.......L.S.Y.LD.G--..........HK......G...Y................D...E.....K.....C.....T...
TaPDIL2-1 ---------------------......AV..S.....E...DS....E...........................S...QDEG--...........TS.....G..................E.ES..E.....K...........S...
TaPDIL2-2 ---------------------......AV..S.....E...DS....E...........................S...QDEG--...........TS.....G..................E.ES..E.....K.......N-------
TaPDIL2-3 ---------------------......AV..S.........DS....E.................H.......L.S.Y.QDEG--..........HTA.....G....................D...E.....K.....C.....S...
TaPDIL2-4 GLLRDRLDGKTSGGSSGXTSGGXSEKINE.NES.E.NSSN.D.L.CQSKDLWI...F........K...EWKRA.RT-------------------------------------------------------------------------
P5(Human) QLVKDRLGGRSGGYSSGKQGRSDSSS---KKD.IE..DDS.DKN...SEDVWM............N.E.EWAAA.SEV.EQTKGK.KL.AV..TVNQV..SR.GIR....I.I.Q..-ESPV..D...TRS.I.SRALDLFSDNAPPPEL
AtPDIL2-2 ALLKDRLDGKTSG--TKNGGGSSE.KKSE..AS.E.NSSN.D.L.TESKELWI...F........K...EWK.A.NNL.GK----.KLGHVNC.AEQSIKSRFK.Q....ILV.GSDKSSPVP.E.A.SASAIES.AL.QLESNAGPAEV
AtPDIL2-3 GLLSDRLEGKS-----KPTGGGSKEKKSE..AS.E.NASN.DDL.IESNELWI...F........K...EWKRA.KNLQGK----.KLGHVNC.VEQSIMSRFK.Q....ILV.GPDKSSPYP.E.A.SASAIES.AS.LVES.AGPVEV
OsPDIL2-3 ALLRDRLNGKTSAGSGGKKSGGSSEK-.E..ASIE.NSQN.DKL.TKSKDLWI...F........K...EWK.A.KNL.GQ----.KLGHV.C.AEKS.MS..K.E....ILV.GADKESPFP.Q.A.VASAIES.AL.QLEANAAPPEV
OsPDIL2-3a ALLRDRLNGKTSAGSGGKKSGGSSEK-.E..ASIE.NSQN.DKL.TKSKDLWI...F........K...EWK.A.KNL.GQ----.KLGHV.C.AEKS.MS..K.E....ILV.GADKESPFP.Q.A.VASAIES.AL.QLEANAAPPEV
OsPDIL2-3b ALLRDRLNGKTSAGSGGKKSGGSSEK-.E..ASIE.NSQN.DKL.TKSKDLWI...F........K...EWK.A.KNL.GQ----.KLGHV.C.AEKS.MS..K.E....ILV.GADKESPFP.Q.A.VASAIES.AL.QLEANAAPPEV
TaPDIL2-5 GLLRDRLDGKTSGGSSGKTSGGSSEKKHE.NES.E.NSSN.D.L.VKSKDLWI...F........K...EWKRA.KNL.GQ----.KLGHV.C.SDKS.MS..K.E....ILV.GADKESPFP.Q.A.AASAIES.AL.QLEANAAPPEV
ao
a
TRX
TRX
B
193
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
P5(Alfalfa LTSEAGIVEDLDELVKEFVAANDEEKKAVFARIEEEVKKLEGSAS-RYGKIYLKVSKKYLEKGSDYAKNEIQRLERLLEKSISPAKADELTLKKNILSTYA-------------------------------------------------
AtPDIL2-1 ...K.....S..A....L...SED.....LS.....AST.K..TT-....L...LA.S.I.......SK.TE..G.V.G.....V........R...T.FVASS----------------------------------------------
OsPDIL2-1 .........S.AP.....LG.ANDKR.EALSKM..D.A..T.P.A-K.....VNSA..IM....E.T.K.SE..Q.M.......S....FVI.......FSS------------------------------------------------
OsPDIL2-1a .........S.AP.....LG.ANDKR.EALSKM..D.A..T.P.AN......VNSA..IM....E.T.K.SE..Q.M.......S....FVI.......FSS------------------------------------------------
OsPDIL2-2 ......RIAS..A.A...LG.ANDKR.EILSNM....V..S...A-KH..V.IAIA..I.D..H..T.K.TE....M.......S....FII...V...FSS------------------------------------------------
OsPDIL2-2a ......RIAS..A.A...LG.ANDKR.EILSNM....V..S...A-K-------------------------------------------------------------------------------------------------------
TaPDIL2-1 ......L.AS..A.....HS.A.DKR.EILSK....AA..S.P.V-KH....VN.A..I.Q.....T.K.TE..H.........S....FAI......AFSS------------------------------------------------
TaPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaPDIL2-3 .........S.AP.....LG.ANDKR.EALSKM..D.A..T.P.A-K.....VNSA..IM....E.T.K.SE..Q.M.......S....FVI.......FSS------------------------------------------------
TaPDIL2-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
P5(Human) .EIINEDIAKRTCEEHQLCVVAVLPHILD-----TGAAGRNSYLEVLLKLADKYKK.MWGWLWTEAGAQSELETALGIGGFGY..M.AINAR.MKFALLKGSFSEQGINEFLRELSFGRGSTAPVGGGAFPTIVEREPWDGRDGELPVED
AtPDIL2-2 TELTGPD.MEDKCGSAAICFVSFLPDILD-----SKAEGRNKYLEMLLSVADKFKKDP.GFVWVAAG.QPDLEKRVGVGGYGY..MVALNAK.GAYAPLKSGFEVKHLKDFVKEAAKGGKGNLPIDG--TMEIVKTEAWDGKDGEVVDAD
AtPDIL2-3 TELTGPD.MEKKCGSAAICFISFLPDILD-----SKAEGRNKYLEMLLSVAEKFKKQP.SFMWVAAVTQMDLEKRVNVGGYGY..MVAMNVK.GVYAPLKSAFELQHLLEFVKDAGTGGKGNVPMNG--TPEIVKTKEWDGKDGELIEED
OsPDIL2-3 SELTGPDAMEEKCASAAICFVSFLPDILD-----SKAEGRNKYLELLLSVAEKFKKSP.SFVWTAAG.QADLEKQVGVGGYGY..MVALNVK.GAYAPLRSAFQLDEITEFVKEAGRGGKGNLPLDG--TPTIVQSEPWDGKDGEVIEED
OsPDIL2-3a SELTGPVSFRDINK.ILLWRQWSGIDVLTRWFGFCRTPWKRNVLLLPFALYLSFQISWI---------QRPKEETST.SCY---YLLLRNLKR--------------VHTVLSGQLLGSKLILRSKL--ELVVMAIQRWLLST-------
OsPDIL2-3b SELTGPDAMEEKCASAAICFVSFLPDILD-----SKAEGRNKYLELLLSVAEKFKKSP.--------RQDSTK.VVDFIPFYLSFLVLLCL.R---------------------------------------------------------
TaPDIL2-5 SELTSAD.MEEKCASAAICFVSFLPDILD-----SKAEGRNKYLELLLSVAEKFKKSP.SFVWAGAG.QADLEKQVGVGGYGY..MVALNVK.GAYAPLRSAFELAEITEFVKEAGRGGKGNLPLEG--APTVVQSEPWDGKDGEVIEED
460 470
. . . . | . . . . | . . . . | . . . . | . .
P5(Alfalfa ----------------------
AtPDIL2-1 ----------------------
OsPDIL2-1 ----------------------
OsPDIL2-1a ----------------------
OsPDIL2-2 ----------------------
OsPDIL2-2a ----------------------
TaPDIL2-1 ----------------------
TaPDIL2-2 ----------------------
TaPDIL2-3 ----------------------
TaPDIL2-4 ----------------------
P5(Human) DIDLSDVELDDLG-----KDEL
AtPDIL2-2 EFSLEDLMGNDDEASTESKDDL
AtPDIL2-3 EFSLDELMGGDD--AVGSKDEL
OsPDIL2-3 EFSLEELMADNS----PVNDEL
OsPDIL2-3a ----------------------
OsPDIL2-3b ----------------------
TaPDIL2-5 EFSLEELMADSS----APNDEL
b
D
c
194
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
ERp18 ----------------------------------------------------------------------------------------------METRPRLGATCLLGFSFLLLVISSDGHNGLG-------------KGFGDHIHWRTL
AtPDIL5-1 -------------------------------------------------------------------------------------------------MTLGARLVAPMIIL..FIP---IELVKA------------------EVITL.P
AtPDIL5-2 -----------------------------------------------------------------------------------------------------MRSLK.LLCWISFLTL.ISISAS----------SDDQFTLDGTVLEL.D
AtPDIL5-3 MVSSTKLKSVDFYRKIPRDLTEASLSGAGLSIVAALFMMFLFGMELSSYLEVNTTTAVIVDKSSDGDFLRIDFNISFPALSCEFASVDVSDVLGTNRLNITKTVRKFPIDPH.RSTGAEF.S..ALHNINHG--EETKEE.P.GAIPL.S
AtPDIL5-4 MVSTSKIKSVDFYRKIPRDLTEASLSGAGLSIIAALSMIFLFGMELNNYLAVSTSTSVIVDRSADGDFLRLDFNISFPSLSCEFASVDVSDVLGTNRLNVTKTIRKFSIDSNMRPTG.EF.A.EVLSLINHG--DETGEEIVEDSVPL.G
OsPDIL5-1 -------------------------------------------------------------------------------------------------MDLAPGRRARLLVA.A..---VLVALAAR--------------S.AEVITL.E
OsPDIL5-1a -------------------------------------------------------------------------------------------------MDLAPGRRARLLVA.A..---VLVALAAR--------------S.AEVITL.E
OsPDIL5-2 --------------------------------------------------------------------------------------------MAAT.TRP.PLLL..LLPP...LLL.FHAAAAA---------AAEEFPRDGRVIELDE
OsPDIL5-3 --------------------------------------------------------------------------------------------MGKP.L.PVVVVVV.LLLVVV.PATTC.ADAG.GG-----EAEEFQIPRDGRVLELDD
OsPDIL5-4 -----------------------------------------------------------------------------------------MSVMFW.QLNITKTVRKYSIDRN.VPTG.EF.P.PIPTVSKHG--DDVEENHD.GSVPLSS
OsPDIL5-4a MISSSKLKSVDFYRKIPRDLTEASLSGAGLSIVAALAMVFLFGMELSNYLAVNTSTSVIVDRSSDGEFLRIDFNLSFPALSCEFASVDVSDVLGTNRLNITKTVRKYSIDRN.VPTG.EF.P.PIPTVSKHG--DDVEENHD.GSVPLSS
TaPDIL5-1 -------------------------------------------------------------------------------------------------MDLGAPARRRLPIR....SLTVLVVLTAR--------------SSAEVITL.E
TaPDIL5-2 ---------------------------------------------------------------------------------------------------MDP.RRSRLPTH....AVTLLAALAAR--------------S.AEVITL.E
TaPDIL5-3 -------------------------------------------------------------------------------------------------MDLGAPARRRLPIR....SLTVLVVLTAR--------------SSAEVITL.E
TaPDIL5-4 -------------------------------------------------------------------------------------------------MDLAPGRRARLLVA.A..---VLVALAAR--------------S.AEVITL.E
TaPDIL5-5 -------------------------------------------------------------------------------------------------MATPQIYRKTLLPV...LAAAALYPAAAD---------------..EVLAL.E
TaPDIL5-6 -------------------------------------------------------------------------------------------------MAICKVWIS.LLALAVVLSAPAARAEEA-------AAAEEAAAAPEAVLTLHA
TaPDIL5-7 -------------------------------------------------------------------------------------------------MAICK.WIS.LLALAVVLSAPAARAEEA-------AAAAEEAAAPEAVLTLHA
TaPDIL5-8 --------------------------------------------------------------------------------------------MAPP---PPPPPLP.LLLL.P.LLAPFSATA-----------AAEEFPRDGKVIDLDD
TaPDIL5-9 --------------------------------------------------------------------------------------------MAPP---PPP--LP.LLLP.-.LLAAFS--------------AAEEFPRDGKVIDLDD
TaPDIL5-10 -------------------------------------------------------------------------------------------------------MRPAILAA...LLAAAASPAAALY------------SA.SPVLQLNP
TaPDIL5-11 ---------------------------------------------------------------------------------------------------MAVDKQR.LPL.V.ALVTAC--LVS.GE-----EPARFQIPRDGSVVELDE
TaPDIL5-12 --------------------------------------------------------------------------------------------MAAT.TRP.PLLL..LLPP...LLL.FHAAAAA---------AAEEFPRDGRVIELDE
TaPDIL5-13 MISSSKLKSVDFYRKIPRDLTEASLSGAGLSIVAALAMVFLFGMELSNYLAVNTSTSVIVDRSSDGEFLRIDFNLSFPALSCEFASVDVSDVLGTNRLNITKTVRKYSIDRN.VPTG.EF.P.PIPTVSKHG--DDVEENHD.GSVPLSS
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
ERp18 EDGKKEAAASGLPLMVIIHKSWCGACKALKPKFAESTEISELS------HNFVMVNLEDE---EEPKDEDFSPDGGYIPRILFLDPSGKVHP-----EIINENGNPSYKYFYVSAEQVVQGMKEAQERLTGDAFRKKHLEDEL-------
AtPDIL5-1 .TFSDKIKEKDTAWF.KFCVP..KH..K.GNLWEDLGKAM.GD------DEIEVGEVDCG--TSRAVCTKVEIHS--Y.TFMLFYNGEE---------VSKYK.KRDVESLKAFVVEETEK----------AAEKAQLEDKEL-------
AtPDIL5-2 SNFDSAIST-FDCIF.DFYAP...H..R.N.ELDAAAP.LAKLK-----QPI.IAK.NAD--KYSRLARKIEI.A--F.TLMLYNHGVP----------MEYY.PRKADLLVRYLKKF.A---PDVAVLESDSTVKEFVEDAGTFFPVFI
AtPDIL5-3 ASFEALSHH-FPI.V.NFNAP..YWSNR...SWEKAAN.IKQRYDPEADGRVLLG.VDCT--E.PALCKRNHIQ.--Y.S.RIFRKGSDLREDHGHH.HESYY.DRDTDSIVKMV.GL.APIHPETHKVALDGKSNDTVKHLKK-GPVTG
AtPDIL5-4 RNFDTFTHQ-FPI.V.NFYAP..YW.NL...SWEKAAKQIKERYDPEMDGRVILAKVDCT--Q.GDLCRRNHIQ.--Y.S.RIFRKGSDLKDDNAHHDHESYY.DRDTESLVKMVVSL.EPIHLEPHNLALEDKSDNSSRTLKK-APSTG
OsPDIL5-1 .TFSDKIKEKDTVWF.KFCVP..KH..N.GTLWEDLGKVM.GA------DEIEIGQVDCG--VSK.VCSKVDIHS--Y.TFKVFYEGEE---------VAKYK.PRNVESLKNFVSDEAE-------------KAGEAKLQDS-------
OsPDIL5-1a .TFSDKIKEKDTVWF.KFCVP..KH..N.GTLWEDLGKVM.GA------DEIEIGQVDCG--VSK.VCSKVDIHS--Y.TFKVFYEGEE---------VAKYK.-----NLRL-------------------------------------
OsPDIL5-2 SSFEAALG.-IDY.F.DFYAP...H..R.A.ELD.AAPVLAGLS-----EPIIVAKVNAD--KYRKLGSKYGV..--F.TLMLFIHGVP----------.EYT.SRKADLLVRNLNKF.A---PDVSILESDSAIKSFVENAGTSFPMFI
OsPDIL5-3 GNFDAAVR.-AGL.F.DFYAP...H..R.A.QLD.AAPVLAGLS-----TPI.VAKVNAD--KYKKLGSKYGV..--F.TLMLF.HGTP----------TEYT.SRKADLLVENLKKL.A---PDVSVLESDSAIKSFVEDAGMGFPLFL
OsPDIL5-4 RNFDSYSHQ-YPV.V.NFYAP..YWSNR...SWEKTAK.MRERYDPEMDGRIILAKVDCT--E.IDLCRRHHIQ.--Y.S.RIFRKGSDLKENQGHHDHESYY.DRDTESLVAAM.TY.ANIPKDAHVLALEDKSNKTVDPAKRPAPLTS
OsPDIL5-4a RNFDSYSHQ-YPV.V.NFYAP..YWSNR...SWEKTAK.MRERYDPEMDGRIILAKVDCT--E.IDLCRRHHIQ.--Y.S.RIFRKGSDLKENQGHHDHESYY.DRDTESLVAAM.TY.ANIPKDAHVLALEDKSNKTVDPAKRPAPLTS
TaPDIL5-1 .TFSDKIKEKDTVWF.QFCVP..KH..N.GTLWEDLGKVM.GA------DEIEIGQVDCG--VSK.VCSKVDIHS--Y.TFKVFYEGEE---------VVKYK.PKDVESLKNFVLNEAE------------KASEENLN----------
TaPDIL5-2 .TFSDKIKEKDTVWF.QFCVP..KH..S.GTLWEDLGKVI.GT------DEIEIGKVDCG--ASK.VCSKVDIHS--Y.TFKVFYDGEE---------VAKYK.PRDVEALKTFVLKEAE------------KAGEVRLEDEL-------
TaPDIL5-3 .TFSDKIKEKDTVWF.QFCVP..KH..N.GTLWEDLGKVM.GA------DEIEIGQVDCG--VSK.VCSKVDIHS--Y.TFKVFYEGEE---------VVKYK.PRDVDR----------------------------------------
TaPDIL5-4 .TFSDKIKEKDTVWF.KFCVP..KH..N.GTLWEDLGKVM.GA------DEIEIGQVDCG--VSK.VCSKVDIHS--Y.TFKVFYEGEE---------VAKYK.PRNVESLKNFVSDEAE-------------KAGEAKLQDS-------
TaPDIL5-5 STFE..VGQ-DRGAL.EFYAP...H..K.A.EYEKLAASFKKA------KSVLIAKVDCD--EHKSVCSKYGVS.--Y.T.QWFPKGSLE--------PKKYE.QRTAEALTEYVNSEAA---TNVKIAAVPSSVVVLTEET--------
TaPDIL5-6 DNFDDAI.K-HPFIL.EFYAP...H..S.A.EYDKAAQLLSKHD-----PAI.LAKVDANDEKNK.LAGKYEVQ.--F.TLKIFRNG..N--------.QEYK.PREAEGIVEYLKKQ.G---PASKEIK-APEDATYLEDGKIHIVGVF
TaPDIL5-7 DNFDDAI.K-HPFIL.EFYAP...H..S.A.EYEKAAQLLSKHD-----PAI.LAKVDANDEKNK.LAGKYEVQ.--F.TLKIFRNGX.N--------.QEYK.PREAEGIVEYLKKQ.G---PASRRSR-HLKMPLTLKNARSTLLVFS
TaPDIL5-8 SNFEAALSS-IDF.F.DFYAP...H..R.A.ELD.AAPVLAGLS-----EPIMVAKVNAD--KYRKLGSKYGV..--F.TLMLFIHGVP----------.EYT.SRKADLLVRNLKKF.A---PDVSTLESDSAIKSFVENAGTSFPMFI
TaPDIL5-9 SNFEAALSS-IDF.F.DFYAP...H..R.A.ELD.AAPVLAGLS-----EPIMVAKVNAD--KYRKLGSKYGV..--F.TLMLFIHGVP----------.EYT.SRKADLLVRNLKKF.A---PDVSTLESDSAIKSFVENAGTSFPMFI
TaPDIL5-10 NNF..VLN.-NGVVL.EFFAP...H..Q.T.IWEKAAGVLKG--------VATVAA.DAD--AHKELAQQYGIR.--F.T.KVFL.GKP---------PVDYE.ARDV.PIVNF.LSQ.K---GLLRDRLDGKTSGGFSGKT--------
TaPDIL5-11 GNFEAAV..-VDF.F.DF.AP...H..R.S.QLD.AAPVLAGLS-----TPI.VAKVNA.--KYKKLRSKYGV..--F.TLMLF.HGVP----------TEYT.SRKAGQLVE.LRKL.A---PDVSVLKSDAAIKSFVQEAGVGFPLFI
TaPDIL5-12 SSFEAALG.-IDY.F.DFYAP...H..R.A.ELD.AAPVLAGLS-----EPIIVAKVNAD--KYRKLGSKYGV..--F.TLMLFIHGVP----------.EYT.SREADLLVRNLNKF.A---PDVSILESDSAIKSFVENAGTSFPMFI
TaPDIL5-13 RNFDSYSHQ-YPV.V.NFYAP..YWSNR...SWEKTAK.MRERYDPEMDGRIILAKVDCT--E.IDLCRRHHIQ.--Y.S.RIFRKGSDLKENQGHHDHESYY.DRDTESLVAAM.TY.ANIPKDAHVLALEDKSNKTVDPAKRPAPLTS
aTRX
C
195
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
ERp18 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
AtPDIL5-2 GFGLNESIISGLGRKYKKKAWF.VS.EVS..TMVSYDFDKAPALVANHPTYNEHSVFYGPFED-GFLEEFVKQSFLPLILPINHDTLKLLKDDERKIVLTIVEDE-THESLEKLYKALRAAAHANRDLVFGYVGVKQFEEFVDSFHVDKK
AtPDIL5-3 GCRVEGYVRVKKVPGNLVISAHSGAHSFDSSQMNMSHVVSHFSFGRMISPRLLTDMKRLLPYLGLSHDRLDGKAFINQH-EFGANVTIEHYLQTVKTEVITRRSGQEHSLIEEYEYTAHSSVAQT-----YYLPVAKFHFELSPMQILIT
AtPDIL5-4 GCRVEGYMRVKKVPGNLMVSARSGSHSFDSSQMNMSHVVNHLSFGRRIMPQKFSEFKRLSPYLGLSHDRLDGRSFINQR-DLGPNVTIEHYLQIVKTEVVKS-NGQ--ALVEAYEYTAHSSVAHS-----YYLPVAKFHFELSPMQVLIT
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-2 GFGVNESLIAGYGGKYKKRAWF.VA.DFS..FMVTYDFDKVPALVSLHPKYKEQSVFYGPFEG-SFLEDFIRQSLLPLTVPINTETLKMLDDDDRKVVLAILEDD-SDETSSQLVKVLRSAANANRDLVFGYVGIKQWDEFVETFDISKS
OsPDIL5-3 GFGVDESLIVEYGAKYKNRAWFSVA.DFS..MMVFYDFDKVPALVSVNPKYREQSIFYGPFDDGAFLEDFIRNSLLPLVVPMNRETVKMLNDDGRKVVLMILQDDESDENSPRLIKVLRSAASANRDLVFGYVGVNQWEEFTETF-DVKS
OsPDIL5-4 GCRIEGFVRVKKVPGSVVISARSGSHSFDPSQINVSHYVTQFSFGKRLSAKMFNELKRLTPYVGGHHDRLAGQSYIVKHGDVNANVTIEHYLQIVKTELVTLRSSKELKLVEEYEYTAHSSLVHS-----FYVPVVKFHFEPSPMQVLVT
OsPDIL5-4a GCRIEGFVRVKKVPGSVVISARSGSHSFDPSQINVSHYVTQFSFGKRLSAKMFNELKRLTPYVGGHHDRLAGQSYIVKHGDVNANVTIEHYLQIVKTELVTLRSSKELKLVEEYEYTAHSSLVHS-----FYVPVVKFHFEPSPMQVLVT
TaPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-5 ---FDS-----VVLDETKDVLGE.YAPWVVTAR--VLLRYMRRWPLVSSKMRGV------------------------------------------------------------------------------------------------
TaPDIL5-6 TEFSGTEFTNFLEV.EKLRSDYD.GHTVHANH.PRGDAAVERPLVRLFKPFDELVVDSKDFDV-SALEKFIEASSTPKVVTFDKNPDNHP---------YLLKSSRAMLPGHALFELLHWTG----------------------------
TaPDIL5-7 LNSAALSLQNFLEV.EKLRPEYD.GHTVHCNIPPRG------------------------------------------------------------------------------------------------------------------
TaPDIL5-8 GFGVNESLIAEYGGKYKKRAWF.VA.DFS..WMATYDFNKIPALVAVHPKYNEQSVFYGPFEG-RFLEDFVRQSLLPLTVPINTETLKLLDDDDRKVVLAILEDD-SDVNSTQLVKILRSAAHANRDLVFGYVGVKQWEEFVETFDVSKS
TaPDIL5-9 GFGVNDSLIAEYGGKYKKRAWF.VA.DFS..WMATYDFNKIPALVAVHPKYNEQSVFYGPFEG-RFLEDL--------------------------------------------------------------------------------
TaPDIL5-10 ---SGGXSEKKHEPNESVELNFEVLDELVRQSKDLWIVEFFAPWVWALQEIGS-------------------------------------------------------------------------------------------------
TaPDIL5-11 GFGVDESSIAEYGARYKKKAWFSTA.DFS..LMAVYDFDKIPALVSLNPKYNEQSVFYGPFEG-TFLEDFIRQSLLPMTVPINAETVKMLKDDDRKVVLAVLQDD-SDETSMRLIKILRSAANANHDLVFGYVGVNQWEEFTEPFHDSKS
TaPDIL5-12 GFGVNESLIAGYGGKYKKRAWF.VA.DFS..FMVTYDFDKVPALVSLHPKYKEQSVFYGPFEG-SFLEDFIRQSLLPLTVPINTETLKMLDDDDRKVVLAILEDD-SDETSSQLVKVLRSAANANRDLVFGYVGIKQWDEFVETFDISKS
TaPDIL5-13 GCRIEGFVRVKKVPGSVVISARSGSHSFDPSQINVSHYVTQFSFGKRLSAKMFNELKRLTPYVGGHHDRLAGQSYIVKHGDVNANVTIEHYLQIVKTELVTLRSSKELKLVEEYEYTAHSSLVHS-----FYVPVVKFHFEPSPMQVLVT
460 470 480 490 500 510 520 530 540 550 560 570
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .
ERp18 ----------------------------------------------------------------------------------------------------------------
AtPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------
AtPDIL5-2 TNLPKIVVWDGDEEYDQVTGIETITQEEDHLTQVSRFLEGYREGRTEKKKINGPSFMGFINSMIGIRSVYILVFLVAVIMMLRSLGQVEEPTGVRTATAVRERVDQATTVPEDESSEHKPSDKKED
AtPDIL5-3 ENPKSFSHFITNLCAIIGGVFTVAGILDSIFHNTVRLVKKVELGKNI-------------------------------------------------------------------------------
AtPDIL5-4 ENSKSFSHFITNVCAIIGGVFTVAGILDSILHHSMTLMKKIELGKNF-------------------------------------------------------------------------------
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-2 SQLPKLIVWDRNEEYEVVEGSEKLE-EGDQASQISQFLEGYRAGRTTKKKVSGPSFMGFLNSLVSLNSLYILICVFALLGVMIYFTGQ--DDTPQVRRAHEE------------------------
OsPDIL5-3 SELPTMIVWDKKEEYEIVEGSERLE-EGDYGSQISRFLEGYRAGRTIKKKVGDR-----SPTLLGVNAVYILVFLVAVLVLLMYFSGQGEEDQRPRQRAHED------------------------
OsPDIL5-4 ELPKSFSHFITNVCAIIGGVFTVAGILDSIFHNTLRLVKKVELGKNI-------------------------------------------------------------------------------
OsPDIL5-4a ELPKSFSHFITNVCAIIGGVFTVAGILDSIFHNTLRLVKKVELGKNI-------------------------------------------------------------------------------
TaPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-2 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-3 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-5 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-6 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-7 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-8 SQLPKLLVWDRNEEYELVEGSEKLE-EGDQASQLSQFLEGYRAGRTIKKKVSGPSFMGFMHSLVSMNSLYILMFVVALLGVMMYFTGQ--DDT-QPRRVHDE------------------------
TaPDIL5-9 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-10 ------------------------------------------------------------------------------------------------------------------------------
TaPDIL5-11 SQLPKLVVWDKDEKYEVVEGLEKLE-EGDHGSQISRFLEAYRAGRTIKKTFGRR-----FPTLLGVNALYILLLLVAVLVVLMFFSGQGEEDRQP-TRAHQE------------------------
TaPDIL5-12 SQLPKLIVWDRNEEYEVVEGSEKLE-EGDQASQISQFLEGYRAGRTTKKKVSGPSFMGFLNSLVSLNSLYILICVFALLGVMIYFTGQ--DDTPQVRRAHEE------------------------
TaPDIL5-13 ELPKSFSHFITNVCAIIGGVFTVAGILDSIFHNTLRLVKKVELGKNI-------------------------------------------------------------------------------
196
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
HsQSOXL MRRCNSGSGPPPSLLLLLLWLLAVPGANAAPRSALYSPSDPLTLLQADTVRGAVLG---------------------SRSAWAVEFFASWCGHCIAFAPTWKALAEDVKAWRPALYLAALDCAEETNSAVCRDFNIPGFPTVRFFKAFTK
AtQSOX1 ------M.LIHLF...G..S.E.AASFSPG-SRSILR------DIGSNV------ADQKDNAIELNATNFDSVFQDSPAKYAVL....H..PA.RNYK.HYEKV.RLFNGADAVYPGVV.MTRVDCAIKMNVKLCDKFSINHYPMLFWAP
AtQSOX2 ------M.LVHLL.FAG.V--I.ASSSSPG-SRLILR------EIS----------DQKDKAVELNTTNFDSVLKDTPAKYAV.....H..PA.RNYK.HYEKV.RLFNGPDAIHPGIV.MTRVDCAMKTNTKLCDKFSVSHYPMLFWGP
OsQSOXL1 ------MAAAAVARRVV.VLV..AASLA....G.AAR------S.GGREGP.E---VDADAAVDLNATNFDAFLKASLEPWAV.....H..PA.RNYK.HYEKV.KLFNGRDA.HPGLI.MARVDCA.K.NI.LCNRFSVDHYP.LLWGP
OsQSOXL1a ------MAAAAVARRVV.VLV..AASLA....G.AAR------S.GGREGP.E---VDADAAVDLNATNFDAFLKASLEPWAV.....H..PA.RNYK.HYEKV.KLFNGRDA.HPGLI.MARVDCA.K.NI.LCNRFSVDHYP.LLWGP
TaQsOXL1 ------MAPAAAW---V..LV...ASSL.G-.AGALR------S.GVGDGA..GAAAQGDYAVDLNATSFDAFLTASREQCGV.....H..PA.RNYK.HYEKV.KLFNGPDA.HPGRI.MARVDCA.K.NV.LCSRFSVDHYP.LLWGP
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
HsQSOXL NGSGAVFPVAGADVQTLRERLIDALESHHDTWPPACPPLEPAKLEEIDGFFARNNEEYLALIFEKGGSYLGREVALDLSQHKGVAVRRVLNTEANVVRKFGVTDFPSCYLLFRNGSVSRVPVLMESRSFYTAYLQRLSGLTREAAQTTVA
AtQSOX1 PKR---.-.G.SWGPKQEKNE.SVVNEW--------------RTADLLLNWINKQIGSSYGLDDQKLG--NLLSN-ISD.EQISQAIF--------------------------------------------------------------
AtQSOX2 PTK---.-.S.SWEPKKDKSE.LVIDDG--------------RTA.RLLNWINKQIGSSYGLDDQKFKNEHALSN-LTDYNQISQAVY--------------------------------------------------------------
OsQSOXL1 PTK---.-ASAKWDPKQENNE.KLIDDG--------------RTA.RLLKWIN.QMKSSFSLED.KYENENMLPKNASDPEQI.QAIY--------------------------------------------------------------
OsQSOXL1a PTK---.-ASAKWDPKQENNE.KLIDDG--------------RTA.RLLKWIN.QMKSSFSLED.KYENENMLPKNASDPEQI.QAIY--------------------------------------------------------------
TaQsOXL1 PPK---.-ANTKWDRKQEKSD.KLIDDG--------------RTA.RLLKWINKQL.SSFTLDD.KYENENVLPNNASDPKQV.QAIY--------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
HsQSOXL PTTANKIAPTVWKLADRSKIYMADLESALHYILRIEVGRFPVLEGQRLVALKKFVAVLAKYFPGRPLVQNFLHSVNEWLKRQKRNKIPYSFFKTALDDRKEGAV------LAKKVNWIGCQGSEPHFRGFPCSLWVLFHFLTVQAARQNV
AtQSOX1 -----------------------.I.E.TEEAFD.ILAHKAIKSSETSASFIR.LQL.VAHH.S.RCRTGSAEILVNFDDICPSGECS.DQE-SGAK.SLRNFH--ICGKDVPRGYYRF.R..KNET...S.G....M.S.S.RIE----
AtQSOX2 -----------------------.V.E.TAEAFD.ILAHKAIKSSETSASFIR.IQL..AHHLS.RCRKGAAEILVNYDDLCPSGNCS.EK--SGGN.TLGNFP--ICGKDVPRGYYMF.R..KNDT...S.G....M.S.S.RIE----
OsQSOXL1 -----------------------.V.E.TAQA.Q.ILE.KTIKP-KNRDS.IR.LQI.VARH.SKRCRRGSAELLINFDDHWSS.LSLS.QEGSK.LESVAEENHWICGKEVPRGY.LF.R..KSET...S.G....M.S...RIG----
OsQSOXL1a -----------------------.V.E.TAQA.Q.ILE.KTIKP-KNRDS.IR.LQI.VARH.SKRCRRGSAELLINFDDHWSS.LSLS.QEGSK.LESVAEENHWICGKEVPRGY.LF.R..KSET...S.G....M.S...RIG----
TaQsOXL1 -----------------------.V.E.TAHA.Q.XLEHKMIKA-DTRDS.IR.LQI.VAHH.SQECRXGSAXLLINFDDHWPS.LSLR.TDSSK.WESVAADT---------------TKSGKGGAP.SG.LA.K--------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
HsQSOXL DHSQEAAKAKEVLPAIRGYVHYFFGCRDCASHFEQMAAASMHRVGSPNAAVLWLWSSHNRVNARLAGAP----SEDPQFPKVQWPPRELCSACHNERLDVP---VWDVEATLNFLKAHFSPSNIILDFPAAGSAARRDVQNVAAAPELAM
AtQSOX1 -----DGESQFAFT..CDFINN..M.D..RR..HD.CLSVKTPFKKARDIA.....T..K..E..KKDEDSLGTG..K...MI...KQ..PS.YLSSTEKN--ID..HDQVYK...KYYGQKLVSVYKKNGE.VSKEE.IAA.EEMAVPT
AtQSOX2 -----DGESHFAFTT.CDF.NN..M.DE.RL..ND.CLSVKTPFKKARDF...V..T..K..E..LKDEASLGTG..K...II...K...PL.YLSSNQKS--IE..H.HVYK...NYYG.KLVS.YKEKSV.RSKEETVSATEDLTV.T
OsQSOXL1 -----DGESQSTFTS.CDFI.N..I.EE.RK..YE.CSSVSAPFRTARELS.....T..K..M..MKEEKDMGTG..L....T...NQ..PS.YRSSKVTDGAVD.NED.VYQ..VNYYGKKLVSSYKETYMESLQQQEKKIVSEDSSIS
OsQSOXL1a -----DGESQSTFTS.CDFI.N..I.EE.RK..YE.CSSVSAPFRTARELS.....T..K..M..MKEEKDMGTG..L....T...NQ..PS.YRSSKVTDGAVD.NED.VYQ..VNYYGKKLVSSYKETYMESLQQQEKKIVSEDSSIS
TaQsOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
HsQSOXL GALELESRNSTLDPGKPEMMKSPTNTTPHVPAEGPEASRPPKLHPGLRAAPGQEPPEHMAELQRNEQEQPLGQWHLSKRDTGAALLAESRAEKNRLWGPLEVRRVGRSSKQLVDIPEGQLEARAGRGRGQWLQVLGGGFSYLDISLCVGL
AtQSOX1 N..VVP------------------------------------VGAA.AI.LASCAFGAL.CYW.TQ.KNRKYNYNPHYLKRYNSNYMVMNTFS.TESEREKE.-----------------------------------------------
AtQSOX2 N..VVP------------------------------------IGAA.AI.IASCAFGAL.CYW.TQ.KNRKPRRR---------------------------------------------------------------------------
OsQSOXL1 N.ASVP------------------------------------IGAA.GV.IASCTFGAL.CFW.AQ.KNRKQRKNWN-------------------------------------------------------------------------
OsQSOXL1a N.ASVP------------------------------------IGAA.GV.IASCTFGAL.CFW.AQ.KNRKYSH..RSLKKI--------------------------------------------------------------------
TaQsOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .
HsQSOXL YSLSFMGLLAMYTYFQAKIRALKGHAGHPAA
AtQSOX1 -------------------------------
AtQSOX2 -------------------------------
OsQSOXL1 -------------------------------
OsQSOXL1a -------------------------------
TaQsOXL1 -------------------------------
D
a/a’
TRX
ERV1
i ii iii
197
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AtAPR1 MAMSVNVSSSSSSGIINSRFGVSLEPKVSQIGSLRLLDRVHVAPV-SLNLSGKRSSSVKPLN-AEPKTK-DSMIPLAATMVAEIAEEVEVVE------IEDFEELAKKLENASPLEIMDKALEKYGNDIAIAFSGAEDVALIEYAHLTGR
AtAPR2 ..LA.TS..TAI..SSF..S.A.S.S.AL..C.I..S..T.LS---------Q.RY.M....-..SHSRSE.WVTR.S.LI.PEV..-KGG.------V....Q......D............RF.DQ.................R...K
AtAPR3 ..LAI........A.SS.S.PS.-DL..TK.......N.TN.SAA-..S.......-..A..-VQSI..-E.IV------AS.VT.KLD...------V........R................F.........................
OsAPRL1 ..SATASI..H.VALR--------DL.AAR..AVKQQVAAAP.AG-TAAARAQ.ARA.R..RA...ARQPV.ASAA..PAA.PV..DAAAAAVDAPAPAV.Y.A..QE.QG........R..AMF.S..................K....
TaAPRL1 ..SATASI..H.IALR--------DL.AAR..AV.QQVA.VP.GLPATAPK.Q.ARA.R..CA...AR.PV.ASAASSP-..PVE..ASA.A------AV.Y.A..QE.VG........R..DMF.SE.................K....
AtAPR4 -------MEKEILLLLLVIMFLTVADVDA-VRVPFCATKSAKDSIFG------------------LRDQTC.VSGVESDERPRFVAVT.GD.RWLQIALDMIHK-N.CDYV.LLFYASWCPFSRSFRPSFDVI.SLYSSIPHFAIKESSI
AtAPR5 -------MD.RV.ILFVCAIA..CFTSG.ASSPVDFSVCNYEFELFRFD.E----------------------AKCPPSLYPTPPI..DGDS--LDRLMASQHGN.---YMSVLFYASWCPFSRAVRPKFDML.SMFPQIQHLAVEHSQA
AtAPR6 -------MEKKLTLLLLVVVVLFVNLTNATVRVQICPRESAKDYILG------------------FRD.S-------.LHRPGFV--T.GDDRWLQMAADMVDKKN.CDYA.LLFYASWCPFSRLVRPSFDLM.LLYSSVPHFAIEESSV
AtAPR7 -------MNLWV.IFLV.AIAG.CL.SGFAY----VDVCN.EFE.FRSVIE----------------------QKCPRSLYPSPPI..DGDL--LDKLMDANHGN.---YISILFYTSRCPFSRAVRPKFDVL.SMFPHITHLIVEQSQA
OsAPRL2 ----------MRWWPALPLLLLAVAVAGAGDAAPVCTRPSAAEAIVG-------------------SPEACR----SPLRR--PLGVT.GDDAILARAVNLLHA-N.EDFA.VLFYASWCPFSQECRLRFEKLACIFPTIRHLAIEESTV
OsAPRL3 ------MATRLLCWTAL-LLPIIAATAAASPLPEACPVPTAAEEILG-------------------PGGTCT----TLDRRGDPVGVI.GD.VTLAKA.TLLHM-N.DDYI.VLFYASWCPFSQECKPNFEILASLFPSIRHFAFEESSI
OsAPRL3a ------MATRLLCWTAL-LLPIIAATAAASPLPEACPVPTAAEEILG-------------------PGGTCT----TLDRRGDPVGVI.GD.VTLAKA.TLLHM-N.DDYI.VLFYASWCPFSQECKPNFEILASLFPSIRHFAFEESSI
OsAPRL4 ..AATASA.A.A.AAAFLLLPLLAAAATAG--HGVCPRQPAA.A.LPRQAPAAS...SSSSSS--SSSSSCPAAG-SPGHR.HHVGV..GDDFVLQKAVTLVLQ-NREDFV.ILFYASWCPFS.IFRTDFQKL.SFFPTIAHFSFEESRI
OsAPRL5 -------MTRCAVVAAVAAVLLVAGAAAAGG.EEEEAPSTCAR-RGPGFVD--------------ALAS---RC.CIR-IEPSPPV..RGEA--IAKELNLRHRGV---TYSVLFYAAWCPFSSKFRP.FE.L.TMFPQIYHFTVEESSA
OsAPRL6 -------MAARLLAPLLLLLLLAPL.PPAAAAEPEEAR--CPRERLPPFVA--------------AAAAAALRPSCR.SAERCP...INGE.--LVKELSGK..-----CT.VLFYASWCPFSQRMRPVFDDL.SMFPRIKHLAVEQ.NA
TaAPRL2 ----------MRWWPALPLLLLAVAVAGAGDAAPVCTRPSAAEAIVG-------------------SPEACR----SPLRR--PLGVT.GDDAILARAVNLLHA-N.EDFA.VLFYASWCPFSQECRLRFEKLACIFPTIRHLAIEESTV
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AtAPR1 PFRVFSLDTGRLNPETYRFFDAVEKHYGIRIEYMFPDSVEVQGLVRSKGLFSFYEDGHQECCRVRKVRPLRRALKGLKAWITGQRKDQSPGTRSEIPVVQVDPVFEGLDGGVGSLVKWNPVANVEGNDVWNFLRTMDVPVNTLHAAGYIS
AtAPR2 ..................L......Q...........A....A...N..................................................I......................L.....A..............A...Q..V.
AtAPR3 .Y................L..T...............A....A...N................I.............R......................................................................V.
OsAPRL1 .................QL..K...............AG...A...A................A..........R..R...............AA........S....A..A............D.K...T...A......A...Q..V.
TaAPRL1 .................EL..K.......H......EAS...D..................................................AS........S.......A...I........D.K.I.T..............Q..V.
AtAPR4 KPSTL.KYGVHGF.TLLLLNST--MRARY.GTR.LDSL.AFYSD.TGIETLDKTSLERSV---SVPHLGNENNTEPENCPF.WA.-SPENML.Q.TYLALAIVFVLLRLLHLIY-PTLVVFMKFTWRRIAQNM.LE---SLLE.TV.FL.
AtAPR5 LPS...RYGIHSL.SILMVNQT--LNARYHGRKDLISLI.FYEEATGLQPVQYVAE.EPTG---------LN.GD.NLITWLRKGTSIREIFKQDPFL.LSLLFICLQMAILVF-PIAESRMRALWASYVAN.NLGRFGEISQLFNRG.H
AtAPR6 KASTL.KYGVHGF.TIILMNST--MLVVY.GSRTLDSL.AFYTD.TGIETMDERWVERNR---LVPHF----HAEPENCPFPWA.RSPENLL.Q.TYLTLATVFVLLRLLHLI.-PTMVVFVKFTWGR.S-NM.LG---NPLE.TVTMYL
AtAPR7 LPS...RYGIHSL.SILMVNQT--MKMRYHGPKDLASLIQFYKETTGLKPVQYMDE.EPTS---------LDT-D.NLITWLHNGSSIREIAER.PYM.LALMFLSLKLAILIF-PIMGSRLKTLWALYVPH.SLGILGETSQLFGRALH
OsAPRL2 RL.TRYRYGIHGY.TLFLINST--VRVRYHGPRTVKSLAAFYND.SGINPSMDPAV.-------DDNIEPK.DCEQE.CLFWSA.-TPENILQPDTYLTLAASFVILRLLYLF-YP.ITAFVKRTWSRRTL.-------TCLEQGKHKFN
OsAPRL3 RPSII.RYGIHGF.TLFLLNST--MRVRYHGPRTVKSLAAFYRD.SGFDVSMTS.AVLH----SVDGIE.KKDAEQENCPFWWA.-SPEKILQQDTYLALATAFVILRLLYLL-FP.IGSF.KRAWRRHTL.P------NLVGVHEYFFT
OsAPRL3a RPSII.RYGIHGF.TLFLLNST--MRVRYHGPRTVKSLAAFYRD.SG--------------------------APIITCGVWT--------------LSLSISLVLVVFLH--------------FQ.STEQ.------CYVSLNELVF.
OsAPRL4 KP.ML.RYGV.AF.TLFLVNST--MRVRYHGSRTMNSLAMFYKD.TGMNPV.LDAISLERMEE.VNIIENDKKTEQGDSLFMFA.-SPDRLLHQDTCLALASSFVLMRLLCFL-.P.L.ACVKQAWRMQFY------------ELKRLFP
OsAPRL5 MPSL..RYGV.GF.AILLVNETTMVR.W--GPKDLSSL.DFYKETTGFDPIAYFDVD-------------HQDST.DFRPV.PGDRSLRKIAKD.PF.LLAVLFIILKVAAHFV-PIVVSHLKTFLVVRVQN.NLGIRRGSSQLLERALN
OsAPRL6 MPA.L.RYGV.SF.SILIACGP--YA.WPVGSKELDSL.N.YTA.TGQEPIAYLGPRKW-----------SA.RT.STQHVKLWKSSIIEALK..PYLAFSILFICLKILVAFF-P.FFSCIKGIWVQYFRHANLGILAKL.QLLECVPH
TaAPRL2 RL.TRYRYGIHGY.TLFLINST--VRVRYHGPRTVKSLAAFYND.SGINPSMDPAV.-------DDNIEPK.DCEQE.CLFWSA.-TPENILQPDTYLTLAASFVILRLLYLF-YP.ITAFVKRTWSRRTL.-------TCLEQGKHKFN
TRX
E
198
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AtAPR1 IGCEPCTKAVLPGQHEREGRWWWEDAKAKECGLHKGNVKENSD--------DAKVNGESKSAVADIFKSENLVTLSRQGIENLMKLENRKEPWIVVLYAPWCPFCQAMEASYDELADKLAGS--GIKVAKFRADGDQKEFAKQELQLGSF
AtAPR2 .......RP............................I..EDG-----------AADSKPA..QE..E.N.V.A..KG.V...L.......A.L..................I...E....K--.V.........E..............
AtAPR3 .......R.............................I...TNG-------N.TA.VNGTAS.....N...V.N.................A...................F.......G..--.V............D...K.......
OsAPRL1 .......RP............................IDDQGGAAAAAAHKAGGA..NGSAGAP...E.SGV.S.T.A.V...LR..S.A...L..................L...ER.G.AGG.V..G......E..A..Q.....Q..
TaAPRL1 .......RP.................T.......N..IDKEGQAPKVGVN----G..SAEASAP...Q.QAI.N.T.P.....LR....A...LT........Y........V...E..S..--...........E..P..QA....Q..
AtAPR4 RAVQ-------LCM.R.SNLQGGAMNARAWASKSLAT.SIGDSSSSNRRSSSSQ------------------------------------------------------------------------------------------------
AtAPR5 MVDVRRLWLK.SLVKT.N-FHERAKNAQAWAS-SLAS.SLGQTSSDQS------------------------------------------------------------------------------------------------------
AtAPR6 KEP---------CMS--SNLQEGAMNARAWASKSLAT.SIAESSSSSRSVSASQ------------------------------------------------------------------------------------------------
AtAPR7 MIDVRRLWIK.RLTKT.N-FQERAKN-------ALAS.SLGKSSSQSA------------------------------------------------------------------------------------------------------
OsAPRL2 ----------RVYPSKQGNLHDGARHATAWASKSLAS.SIGEPSTS--------------------------------------------------------------------------------------------------------
OsAPRL3 YLEQARH.FFRLYPSK.GNLQEGARNATAWASKSLAS.SIGEPSTIGRTNSTNELR----------------------------------------------------------------------------------------------
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL4 ---------------------------------SLS------------------------------------------------------------------------------------------------------------------
OsAPRL5 VLDVKRLCSK.RLSNKTRDLRKGASNARAWAS-SFTS.SLGESSSSRQA-----------------------------------------------------------------------------------------------------
OsAPRL6 AVDLRKIWSKCRLMG-------GAMNSRVWAS-SLASMSFGERSSPRAAVL.--------------------------------------------------------------------------------------------------
TaAPRL2 ----------RVYPSKQGNLHDGARHATAWASKSLAS.SIGEPSTS--------------------------------------------------------------------------------------------------------
460 470 480
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .
AtAPR1 PTILVFPKNSSRPIKYPSEKRDVESLTSFLNLVR
AtAPR2 ....L...RAP.A......H...D..M..V..L.
AtAPR3 .......................D..........
OsAPRL1 ....L..SRTA............D..LA.V.SL.
TaAPRL1 ....L..GRTVK...........Q..LA.V.SL.
AtAPR4 ----------------------------------
AtAPR5 ----------------------------------
AtAPR6 ----------------------------------
AtAPR7 ----------------------------------
OsAPRL2 ----------------------------------
OsAPRL3 ----------------------------------
OsAPRL3a ----------------------------------
OsAPRL4 ----------------------------------
OsAPRL5 ----------------------------------
OsAPRL6 ----------------------------------
TaAPRL2 ----------------------------------
TRX
Fig 6.4 Alignment of the putative amino acid sequences of the five groups of PDILs in rice and wheat. A: PDIL1; B: PDIL2; C: PDIL5; D: QSOXL; E: APRL. The putative catalytic sites (TRX), Erv1-like sites, and the C-terminal ER-localisation signal (KDEL) are framed. Identical residues are shaded blue and similar are shaded grey. The domains in secondary structures are indicated with bars.
199
6.3.8 The relationship of PDILs in rice and wheat to those in Arabidopsis and
other organisms
The phylogenetic relationships of the five PDIL groups in the three plant species with
the human PDILs (hPDI; hP5; ERp18) were determined by analysis of alignments of the
putative amino acid sequences, with the Neighbor-Joining program in MEGA 4.0
(http://www.megasoftware.net) (alignment data were shown in Fig 6.4). The
phylogenetic tree of PDIL1s showed Arabidopsis (2 members), rice (4), and wheat (5)
were most closely related to hPDI (Fig 6.5; Table 6.5). The genes encoding TaPDIL1-
1, TaPDIL1-2 and TaPDIL1-3 are homeoalleles located on group 4 chromosomes
(Ciaffi et al., 1999), their putative proteins showing 85% identity to the OsPDIL1-1 that
has been shown to have critical roles in storage protein assortment (Takemoto et al.,
2002), and one of the homeoalleles is shown to be syntenic to the rice gene (Johnson et
al., 2006). Interestingly, the newly identified, highly related TaPDIL1-4 (predicted to
be located on group 4 chromosomes; section 6.3.9, Table 6.6) is 100% identical to
OsPDIL1-1 on amino acid sequence level, suggesting further work is required to see
whether this is also a candidate for storage protein folding and assortment. All five
PDIL1s in wheat were 50-56% identical to AtPDIL1-1 and AtPDIL1-2. TaPDIL1-5
exhibited 60-100% identity to OsPDIL1-2, OsPDIL1-3, and OsPDIL1-3a. The
orthologues of TaPDIL1-6 appear to be OsPDIL1-4, AtPDIL1-3 and AtPDIL1-4, with
50-70% identity.
The phylogenetic tree of PDIL2 group was generated similarly, for wheat (5 members),
rice (7), Arabidopsis (3), and a human PDI (hP5) (alignment data were shown in Fig
6.4) (Fig 6.5B). Three wheat PDIL2s (TaPDI2-1, TaPDIL2-2, TaPDIL2-3) appear to be
orthologues of four rice ones (OsPDIL2-1, OsPDIL2-1a, OsPDIL2-2, OsPDIL2-2a) and
Arabidopsis AtPDIL2-1, the identity between them being 70-100%. The genes
encoding TaPDIL2-1, TaPDIL2-2 and TaPDIL2-3 could thus be duplicates or
homeoalleles. TaPDIL2-4 and TaPDIL2-5 may be orthologues of three members in rice
encoded by one locus (OsPDIL2-3, OsPDIL2-3a, OsPDIL2-3b) (Table 6.1) and two in
Arabidopsis (AtPDIL2-2, AtPDIL2-3), the identity between them being 65-82% (on the
comparable parts). The genes encoding TaPDIL2-4 and TaPDIL2-5 may thus be
duplicates. All active sites in Arabidopsis PDIL2 were the conserved WCGHC (data
200
not shown) and the molecular weights of proteins, calculated as above, ranged from 39
kDa (AtPDIL2-1) to 48 kDa (AtPDIL2-2 and AtPDIL2-3) (Table 6.5).
The phylogenetic tree of PDIL5 generated for wheat (13 members), rice (6),
Arabidopsis (4), human (2) (alignment data were shown in Fig 6.4) (Fig 6.5C) showed
four members in wheat (TaDPIL5-1, TaPDIL5-2, TaPDIL5-3, TaPDIL5-4) were likely
orthologues of OsPDIL5-1 and OsPDIL5-1a encoded by one locus (Table 6.1) in rice
and AtPDIL5-1 in Arabidopsis, the identity between them being 60-72%. The genes
encoding the four members in wheat may thus be alleles or duplicates. No orthologue
of TaPDIL5-5, TaPDIL5-6, TaPDIL5-7 could be identified in rice and Arabidopsis.
TaPDIL5-6 and TaPDIL5-7 shared 82% identity in comparable parts and may be
duplicates or homeoallelic. Three members of wheat (TaPDIL5-8, TaPDIL5-9,
TaPDIL5-12) were likely orthologues of OsPDIL5-2 and AtPDIL5-2 and the identity
between them was 55-80%. The genes encoding these three may again be allelic or
duplicates. No orthologue of TaPDIL5-10 was found in rice and Arabidopsis.
TaPDIL5-11 may be an orthologue of OsPDIL5-3 (73% identity) and AtPDIL5-2 (43%
identity). TaPDIL5-13 may be an orthologue of OsPDIL5-4 and OsPDIL5-4a encoded
by one locus (Table 6.1) and AtPDIL5-3 (54 kDa) and AtPDIL5-4 (54 kDa) the identity
between them being 70-98% (comparable parts). Genes encoding AtPDIL5-3 and
AtPDIL5-4 may be duplicates located on Chromosome 3 and 4, respectively (Houston
et al., 2005). The active sites in three out of four members of PDIL5 in Arabidopsis
were not conserved, with WCKHC (AtPDIL5-1), WCYWS (AtPDIL5-3), and WCYWC
(AtPDIL5-4) being noted. The molecular weights of AtPDIL5-3 and AtPDIL5-4 were
close to the purified 52 kDa PDI (PDI-52) localized in chloroplast.
The phylogenetic tree of QSOXL group (alignment data were shown in Fig 6.4) showed
that TaQSOXL1 may be an orthologue of OsQSOXL and OsQSOXLa encoded by one
locus (Table 6.1) and AtQSOX1 and AtQSOX2 (Fig 6.5D, Table 6.5). The tree based
on alignment of amino acid sequences of APRL members (alignment data were shown
in Fig 6.5) in wheat (2), rice (7), Arabidopsis (7) (data not shown) (Fig 6.5D) suggested
that TaAPRL1 may be orthologous to OsAPRL1 and three members (AtAPR1,
AtAPR2, AtAPR3) in Arabidopsis, the identity between them being 64-81%. The genes
encoding the three members in Arabidopsis appear to be duplicates located on
Chromosome 4
201
(AtAPR1 and AtAPR3) and Chromosome 1 (AtAPR2) (Houston et al., 2005).
TaAPRL2 appears to be an orthologue of four members (OsAPRL2, OsAPRL3,
OsAPRL3a, OSAPRL4) in rice and AtAPR4 and AtAPR6 in Arabidopsis, identity
between them being 40-100%. AtAPR4 and AtAPR6 shared 65% identity and the
genes encoding them are possible duplicates located on Chromosome 1 and 4,
respectively (Houston et al., 2005).
TaPDIL1-2
TaPDIL1-3
TaPDIL1-1
OsPDIL1-1
TaPDIL1-4
AtPDIL1-1
AtPDIL1-2
OsPDIL1-2
OsPDIL1-3
OsPDIL1-3a
TaPDIL1-5
hPDI
AtPDIL1-5
AtPDIL1-6
OsPDIL1-5
AtPDIL1-3
AtPDIL1-4
OsPDIL1-4
TaPDIL1-6
100
100
100
75
100
100
100
100
100
100
47100
100
84
100A
202
OsPDIL2-1
OsPDIL2-1a
TaPDIL2-3
TaPDIL2-1
TaPDIL2-2
OsPDIL2-2
OsPDIL2-2a
P5(Alfalfa
AtPDIL2-1
P5(Human)
AtPDIL2-2
AtPDIL2-3
TaPDIL2-4
TaPDIL2-5
OsPDIL2-3a
OsPDIL2-3
OsPDIL2-3b100
85
100
77
100
100
83
100
100
62
93
100
B
C OsPDIL5-1
TaPDIL5-4
OsPDIL5-1a
TaPDIL5-1
TaPDIL5-3
TaPDIL5-2
AtPDIL5-1
AtPDIL5-3
AtPDIL5-4
TaPDIL5-13
OsPDIL5-4
OsPDIL5-4a
TaPDIL5-5
TaPDIL5-10
TaPDIL5-6
TaPDIL5-7
AtPDIL5-2
OsPDIL5-3
TaPDIL5-11
OsPDIL5-2
TaPDIL5-12
TaPDIL5-8
TaPDIL5-9
ERp18
53
100
64
100
100
99
83
99
55
83
100
68
95
83
87100
99
98
203
OsQSOXL1
OsQSOXL1a
TaQsOXL1
AtQSOX1
AtQSOX2
HsQSOXL
100
100
59D
OsAPRL2
TaAPRL2
OsAPRL3
OsAPRL3a
OsAPRL4
AtAPR4
AtAPR6
OsAPRL6
AtAPR5
AtAPR7
OsAPRL5
OsAPRL1
TaAPRL1
AtAPR2
AtAPR1
AtAPR391
78
100
100
100
55
100
55
79
59
69
E
Fig 6.5 Phylogenetic analysis of PDILs in Arabidopsis, rice, and wheat. A: PDIL1s; B: PDIL2s; C: PDIL5s; D: QSOXLs and APRLs. Trees were generated by Neighbor-Joining of the Bootstrap Test of Phylogeny in MEGA 4.0 program (http://www.megasoftware.net/mega.html), based on alignments of respective PDIL group members in the three species by ‘ClustalW Multiple Alignment’ application in Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) (Alignment figures were shown in Fig 6.4).
204
Table 6.5 PDIL members in wheat and their potential orthologues in other plants*
Wheat Rice Arabidopsis (MW: kDa)
Others Functions (see section 1.5)
TaPDIL1-1 OsPDIL1-1 AtPDIL1-1 (56) AtPDIL1-2 (56)
GmPDIL-1 GmPDIL-2 (Soybean) hPDI (human)
•Segregation of prolamins and pro-glutelins in ER lumen through chaperone activity in rice (Takemoto et al., 2002).
•Proper seed development and regulation of timing of PCD by chaperoning and inhibiting Cys proteases. (Ondzighi et al., 2008).
•Role in the folding of storage protein by both the thiol-oxidoreductase activity and as molecular chaperone. (Kamauchi et al., 2008; 2007).
•ER-stress response (Lu and Christopher, 2008a).
• Purified 60 kDa PDI (TaDPIL1-6), co-localized with storage proteins in dense protein bodies (Shimoni et al., 1995b).
TaPDIL1-2 TaPDIL1-3 TaPDIL1-4 TaPDIL1-5 OsPDIL1-2,
OsPDIL1-3a, OsPDIL1-3
TaPDIL1-6
OsPDIL1-4
AtPDIL1-3 (64) AtPDIL1-4 (66)
n/a OsPDIL1-5 AtPDIL1-5 (60) AtPDIL1-6 (60)
n/a n/a
TaPDIL2-1 OsPDIL2-1, OsPDIL2-1a, OsPDIL2-2, OsPDIL2-2a
AtPDIL2-1 (39) n/a Function in ovule structure and embryo sac development and pollen tube guidance, through its roles in maturation of secreted or plasma membrane proteins in Arabidopsis (Wang et al., 2008)
TaPDIL2-2
TaPDIL2-3
TaPDIL2-4 OsPDIL2-3, OsPDIL2-3a, OsPDIL2-3b
AtPDIL2-2 (48) AtPDIL2-3 (48)
GmPDIM (Soybean) hP5 (human)
•GmPDIM shown to play a role in the folding of storage protein as both thiol-oxidoreductase and molecular chaperone (Kamauchi et al., 2008; 2007). •ER-stress response (Lu and Christopher, 2008a).
TaPDIL2-5
TaPDIL5-1 OsPDIL5-1, OsPDIL5-1a
AtPDIL5-1 (17)
n/a n/a TaPDIL5-2 TaPDIL5-3 TaPDIL5-4 TaPDIL5-5 n/a n/a n/a n/a TaPDIL5-6 n/a n/a n/a n/a
TaPDIL5-7 n/a n/a n/a n/a TaPDIL5-8 OsPDIL5-2 AtPDIL5-2 (50) n/a n/a TaPDIL5-9 TaPDIL5-10 n/a n/a n/a n/a TaPDIL5-11 OsPDIL5-3 AtPDIL5-2 (50) n/a n/a
TaPDIL5-12 OsPDIL5-2 TaPDIL5-13 OsPDIL5-4,
OsPDIL5-4a AtPDIL5-3 (54) AtPDIL5-4 (54)
n/a n/a
TaQSOXL OsQSOXL1; OsQSOXLa
AtQSOX1; AtQSOX2
n/a n/a
TaAPRL1 OsAPRL1 AtARP1, AtAPR2, AtAPR3
n/a n/a
TaAPRL2 OsAPRL2, OsAPRL3, OsAPRL3a, OsAPRL4
AtAPR4, AtAPR6, AtAPR7
n/a n/a
* The names of PDIL members are: wheat and rice (as given in this study); Arabidopsis (Houston et al., 2005); soybean (Kamauchi et al., 2008); human (Hatahet and Ruddock, 2007).
205
6.3.9 Chromosomal localisation of PDILs in rice and prediction of chromosomal
location of PDILs in wheat
The map of chromosomal locations of the nineteen putative PDIL loci in rice was
generated from Oryza sativa genome view (http://www.ncbi.nlm.nih.gov) with
representative ESTs listed in Table 6.1 (Fig 6.6). The loci were distributed on all
chromosomes except 10. The distance between two loci on the same arm ranged from
200 kb (OsPDIL1-3 to OsPDIL5-3) to 11 Mb (OsPDIL5-3 to OsAPRL3). Genes on the
same chromosome and transcribed in the same direction included OsPDIL5-3 and
OsAPRL3 on chromosome 2 and OsPDIL1-2 and OsPDIL5-2 on chromosome 4, while
the divergently transcribed ones included OsPDIL1-3 and OsPDIL5-3 on chromosome
2, OsPDIL1-5 and OsAPRL2 on 6, and OsPDIL5-4 and OsAPRL1 on 7 (transcription
directions data not shown) .
Fig 6.6 Likely Locations of rice PDILs on chromosomes The map was generated from the Oryza sativa (rice) genome view in the NCBI Map viewer database (http://www.ncbi.nlm.nih.gov) with representative cDNA/ESTs as queries.
The chromosomal locations of the putative wheat PDIL loci were identified
progressively as detailed in chapter 2 (example shown in Fig 2.1). Searching in the
‘landmark or region’ for each of the nineteen rice PDIL loci on the
(http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/) led to identification of five
wheat bin-mapped markers (Table 6.5, Appendix VII). These were entered into
‘Mapped Loci for EST-derived Probes’ query in GrainGenes structured query language
(SQL) database (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi), leading to
identification of cDNA probes used for Southern blotting. The results showed that (i)
206
the bin-mapped marker CNL220BE398523 had been mapped to wheat chromosome 4
(as reported by Johnson et al., 2006); (ii) CSU046BE444859 to chromosome 1 (C-1DS:
3-0.48 1BS:10-0.50-0.84) by two probes (5’ probe: BE444859, 3’ probe: BQ161460);
(iii) UCD078BF201426 to chromosome 6; (iv) NDS021BE500255 to chromosome 1;
(v) KSU035BE403404 to chromosome 1 and 2; and (vi) NDS252BE445181 to
chromosome 5. The wheat PDIL bin-mapping data derived from the rice genome
browser is detailed in Table 6.6.
Table 6.6 Wheat and rice putative PDIL orthologues and their chromosomal locations Rice locus* Chromo-some and position of locus (bp)* PDIL gene
Wheat bin mapped Marker*
Probes for southern blotting*
Chromosome location (cM)*
PDIL gene in wheat
Id(%)**
Os11g09280 Chr11: 4960745-4964540
OsPDIL1-1 CNL220 BE398523
5’BE398523; 3’BQ167770
4AL5-0.66-1.00; C-4BL1-0.71; 4DS1-0.53-0.67
TaPDIl1-1 97.1
TaPDIL1-2 99.7
TaPDIL1-3 97.7 TaPDIL1-4 88.7
Os01g23740 Chr1: 13336110-13339991
OsPDIL2-2
CSU046 BE444859
5’BE444859; 3’ BQ161460
C-1DS: 3-0.48; 1BS:10-0.50-0.84
TaPDIL2-1 99.3
TaPDIL2-2 98.7
TaPDIL2-3 86.7 Os04g35290 Chr4: 21274652-21277663
OsPDIL5-2 UCD078 BF201426
5’ BF201426; 3’ BQ170402
C-6AL: 4-0.55; 6BL: 3-0.36-0.40; 6DL: 6-0.29-0.47
TaPDIL5-8 70.1
Os02g34530 Chr2: 20694520-20696830
OsPDIL5-3
TaPDIL5-9 70.5
TaPDIL5-11 100
TaPDIL5-12 70.1 Os05g47930 Chr5: 27390774-27395793
OsQSOXL NDS021 BE500255
5’ BE500255 3’ CD454155
1DL: 2-0.41-1.00 TaQSOXL 91.1
Os07g32570 Chr7: 19408263-19411558
OsAPRL1
KSU035 BE403404
5’ BE403404 3’ CD373598
1AL: 1-0.17-0.61, C-1BL: 6-0.32; C-2AS: 5-0.78, C-2BS: 1-0.53, C-2DS: 1-0.33
TaAPRL1 97.0
Os12g35640 Chr12: 21632781-1635926
OsAPRL6 NDS252 BE445181
5’ BE445181 3’ CD452815
5AS1-0.40-0.75, 5BS: 4-0.43-0.56
n/a
*Information about loci with position and wheat bin mapped marker is from TIGR Rice Genome Annotation (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/); sequences of probes from EST database (http://www.ncbi.nlm.nih.gov); chromosome location from GrainGenes structured
query language (SQL) database (http://wheat.pw.usda.gov/cgi-bin/westsql/map_locus.cgi). ) The bin-mapped marker CNL220BE398523 was reported to be mapped to chromosome 4 (Johnson et al.,
2006). **Sequence identity of the 5’probe with the corresponding section of sequence of the TA encoding wheat PDIL.
Sequences of 5’ probes were searched in the EST database
(http://www.ncbi.nlm.nih.gov) and aligned with the corresponding wheat TAs for
identity calculation (Table 6.6). The cDNA sequence of BE398523 showed significant
identities (97.1%, 99.7%, 97.7% respectively) with TAs of TaPDIL1-1, TaPDIL1-2,
TaPDIL1-3; and 88.7% identity with TA of TaPDIL1-4. BE444859 showed significant
identities (99.3%, 98.7%, and 86.7 % respectively) with TAs of TaPDIL2-1, TaPDIL2-
2, and TaPDIL2-3 in overlapping sections. BF201426 similarly aligned with
corresponding sections of TAs of TaPDIL5-8, TaPDIL5-9, TaPDIL5-11 and TaPDIL5-
12 (70.1%, 70.5%, 100% and 70.1% identities). BE500225 aligned with TaQSOXL1
207
with 91.1% identity and BE403404 with TaAPRL1 with 97% identity. A map was then
generated from NCBI Map viewer (http://www.ncbi.nlm.nih.gov) using the probes as
queries (Fig 6.7). Three genes (TaPDIL1-1, TaPDIL1-2, and TaPDIL1-3) were
reported to be located on the 4 chromosome groups (Johnson et al., 2006) plus one
(TaPDIL1-4) was also predicted to be located on the chromosome 4. In addition, ten
more genes were located as: TaPDIL2-1, TaPDIL2-2 and TaPDIL2-3 on the short arms
of the chromosome 1B or 1D; TaPDIL5-8, TaPDIL5-9, TaPDIL5-11, TaPDIL5-12 on
the long arms of 6A, 6B, and 6D; TaAPRL on 1A, 2A, 2B, and 2D; and TaQsOXL on
the long arm of 1D. QTLs co-localizing with PDIL family in rice or the corresponding
chromosome in wheat were then identified based on this information.
Fig 6.7 Likely chromosomal locations of wheat PDILs The map was generated from the Oryza sativa (rice) genome view in the NCBI Map viewer database (http://www.ncbi.nlm.nih.gov) with representative cDNA/ESTs as queries.
6.3.10 Identification of quantitative trait loci likely to be associated with PDILs in
rice and wheat
The reported quantitative trait loci associated with rice grain quality or plant
development traits were identified by browsing the Oryza sativa ssp. japonica Group
genome database (http://gramene.org) (Appendix IX). The distribution of quality QTL
accession IDs ranged from two IDs on the chromosome 4 to seventeen on the
chromosome 6. Interestingly, the total protein content QTL (AGFU022) (0-116cM on
chromosome 6) was located on the 3.6Mb region that happended to contain OsPDIL1-5,
208
and consistency viscosity QTL (CQAG19) (17.3cM on chromosome 6) was located on
the 3.9 Mb region that contained the OsAPRL2 gene. Total protein content and
consistency viscosity traits are relevant to the food industry, as introduced in Chapter 1.
The QTLs related to wheat grain quality were identified preliminarily by searching the
integrated wheat science database (http://www.shigen.nig.ac.jp). The search focussed on
chromosomes 1A, 1B, 1D, 2A, 2B, 2D, 6A, 6B and 6D as these contained the PDIL
genes (see above). QTLs related to sedimentation value of flour located on 6AL and
QTLs for endosperm colour were on 2A, with associated marker Xgwm45 (6.1cM) and
on 6B, with associated marker Xgwm193 (13.2cM) (Pozniak et al., 2006). QTL related
to mixograph peak time located on 1DL (Campbell et al., 2001) and that related to
alveograph dough strength located on 1A with associated marker Xfba92 (84cM), 1B
(associated marker Xmta14), and 6B (associated marker Xfbb250). QTL related to
protein content located on 6A with associated marker XE38M60200 (59cM) (Perretant
et al., 2000), and that related to dough rheological properties located on 1AL, 1B, 1D
and 2BL (Ma et al., 2005).
6.3.11 Expression analysis of selected PDILs in different tissues from Arabidopsis
microarray data
Prediction of expression of selected PDILs (PDIL1, PDIL2 and PDIL5 members) in
different tissues and developing seeds in Arabidopsis was conducted using the BAR
(Botany Array Resource) Arabidopsis eFP (Electronic Fluorescent Pictograph) browser
(http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi). Data were obtained by
exploring the microarray data of expression of approximately 22,000 genes in
Arabidopsis thaliana represented on the ATH1 GeneChip from Affymetrix (Winter et
al., 2007). AGI IDs of loci for PDILs in Arabidopsis were used as queries to search the
eFP browser, and expression values were obtained. The data were analysed in
Microsoft Excel (Fig 6.8). Interestingly, the most expressed genes in different tissues
were AtPDIL1-1, AtPDIL1-2, AtPDIL1-3, AtPDIL1-4, AtPDIL2-1, AtPDIL2-3,
AtPDIL5-1, AtPDIL5-2 and AtPDIL5-4, and exhibit the conserved active sites
WCGHC/WCKHC/ WCYWC (Fig 6.4). The less-expressed genes were AtPDIL1-5,
AtPDIL1-6, AtPDIL1-3, and their active sites are not conserved (Fig 6.4). AtPDIL1-1
is the orthologue of TaPDIL1-1, TaPDIL1-2, TaPDIL1-3, and TaPDIL1-4 in wheat. It
209
was selected for the expression analysis in developing seeds. AtPDIL1-1 was expressed
highly (absolute expression >1000) in imbibed seed and developing seed (stages 5, 6,
7). AtPDIL2-1 expressed highly (level >1000) in imbibed and developing seed (stages
3, 4, 5, 6, 7), root, and shoot apex. The expression image of AtPDIL1-1 in developing
seeds was obtained directly in the same database (Fig 6.8) and was very low at seed
stage 3w, increased to the highest level at stage 6 w, and then decreased in seed stage
10.
0
1000
2000
3000
4000
5000
6000
Dry s
eed
Imbib
ed s
eed, 2
4 h
Caulin
e Le
af
Cotyle
don
Root
Flowerin
g
Shoot
Apex
Stem
Mat
ure P
olle
n
Seeds
sta
ge 3
w
Seeds
sta
ge 4
w
Seeds
sta
ge 5
w
Seeds
sta
ge 6
w
Seeds
Sta
ge 7
w
Seeds
Sta
ge 8
w
Seeds
Sta
ge 9
Seeds
Sta
ge 1
0
Veget
ative
Ros
ette
AtPDIL5-4
AtPDIL5-3
AtPDIL5-2
AtPDIL5-1
AtPDIL2-3
AtPDIL2-2
AtPDIL2-1
AtPDIL1-6
AtPDIL1-5
AtPDIL1-4
AtPDIL1-3
AtPDIL1-2
AtPDIL1-1
Fig 6.8 PDIL expression levels in different tissues in Arabidopsis (upper) and expression of AtPDIL1-1 in developing seeds (below). Data obtained from Arabidopsis eFP (Electronic Fluorescent Pictograph) browser database (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cg) (Winter et al., 2007). Data of expression value were transferred to Microsoft Excel for producing the Figure of absolute expression.
AtPDIL1-1
210
6.3.12 Preliminary expression analysis of three homeologous PDI genes in different
tissues and developing seeds in wheat
Preliminary analysis of expression of the wheat homeologues PDIL1-1, PDIL1-2,
PDIL1-3,1, located on 4AL, 4BS, and 4DS and likely to be putative orthologues
(Johnson et al., 2006) of the rice gene related to storage protein assortment (Takemoto
et al., 2002), was conducted by semi-quantitative reverse transcriptase PCR (RT-PCR).
First-strand cDNA synthesized from total RNA from stems, leaves and developing
seeds (6, 10, 14, 18, 25 and 30DAA) was amplified with various primers (allele-specific
primer pairs PDI-A3FPDI-A4R, PDI-B2F/PDI-B3R, PDI-D5F/PDI-D6R, and house-
keeping control ACTIN; Table 2.8) (Johnson and Bhave, 2004a). Amplification of
gDNA was also conducted for comparisons, to check that the RNA had no gDNA
contamination. The experiment was repeated and the fig has two full sets.
The RT-PCR results are shown in Fig 6.9 (first set of samples: panels A, B, C, D;
second set of samples: panels E, F, G, H). The PCR products were ~650bp (TaPDIL1-
1), ~700bp (TaPDIL1-2) and ~750bp (TaPDIL1-3), corresponding to the expected sizes
for cDNA alone (Table 6.7) and much smaller than those expected from gDNA due to
introns (~1800bp for TaPDIL1-1 and TaPDIL1-2, ~1000bp for TaPDIL1-3) (Johnson
and Bhave, 2004b; Ciaffi et al., 2006), confirming the good quality and lack of gDNA
contamination of RNA preparations. Products of simultaneous amplifications with the
ACTIN primer pair were as expected (410bp). Some variation was noted in the two sets
of samples and hence the results cannot be compared between them. However, both
experiments show firstly that all three PDIL1 homeologous genes are expressed and
there is no silencing of them during polyploidy, as has been reported for certain wheat
genes (He et al., 2003), and confirm that PDI is expressed in developing seeds as
previously reported (Grimwade et al., 1996). Since this locus is orthologus to the rice
locus shown to be directly involved in assortment of storage protein subtypes in rice and
deposition of two different types of protein bodies (Takemoto et al., 2002), the wheat
genes may also play such functions during this key event in the developing endosperm.
211
M S L 6 10 14 18 25 30 G
2000bp
1030bp
500bp
100bp
A
M S L 6 10 14 18 25 30 G
2000bp
1030bp
500bp
100bp
B M S L 6 10 14 18 25 30 G
1030bp
500bp
100bp
C
500bp
100bp
M S L 6 10 14 18 25 30
D
700bp 500bp
100bp
M S L 6 10 14 18 25 30
E
800bp 500bp
100bp
M S L 6 10 14 18 25 30
F
700bp 500bp
100bp
M S L 6 10 14 18 25 30
G
500bp
100bp
M S L 6 10 14 18 25 30
H Fig 6.9 RT-PCR products amplified from the first-strand cDNA or genomic DNA of various tissues and developing seeds of wheat. First set (panels A, B, C, D), A: TaPDI L1-1; B: TaPDIL1-2; C: TaPDIL1-3; D: actin gene. Second set (panels E, F, G, H), E: TaPDIL1-1; F: TaPDIL1-2; G: TaPDIL1-3; H: actin gene. M: GeneRuler
TM DNA ladder; S: stem, L: leave, G: genomic DNA, 6-30: developing seeds at
different DAA. The PCR products were separated on 1.0% agarose gels.
212
6.4 Discussion
6.4.1 Rice and wheat PDILs comprise large and diverse families
This study has identified 19 loci encoding 28 putative PDILs in rice, including seven
loci possibly encoding different splice forms. The putative proteins ranged from 22.7
kDa to 59.1 kDa and fell into five groups as reported in Arabidopsis (Houston et al.,
2005), i.e., PDIL1s (6), PDIL2s (7), PDIL5s (6), QSOXL (2) and APRL (7). The gene
structures varied from 3-exon to 15-exon. OsPDIL1-1 is reported to be close to esp2
locus on chromosome 11 (Johnson et al., 2006) and the protein encoded by it may
function in segregation of proglutelin and prolamin polypeptides within the ER lumen
(Takemoto et al., 2002). The 19 loci in rice were dispersed on 11 chromosomes, with
the maximum number on chromosome 2 (four loci) and one each on five different ones.
Some genes may be duplicates dispersed on different chromosomes, i.e., Os11g09280,
Os04g35600 and Os02g34940; Os05g06430 and Os01g23740; Os04g35290 and
Os02g34530; Os06g11740 and Os02g51850.
Twenty-seven TAs encoding putative PDILs in wheat have also been identified from
TIGR Plant Transcript Assemblies database. The size of the 27 PDILs ranged from
14.7 kDa to 63.7 kDa and they fell into the same five groups, i.e., PDIL1 (6), PDIL2
(5), PDIL5 (13), QSOXL (1) and APRL (2). TaPDI1-1, TaPDIL1-2, and TaPDIL1-3
have been reported previously as wPDI1, wPDI2, and wPDI3 (Johnson et al., 2001) or
CPDI-4A, CPDI-4B, and CPDI-4D (Ciaffi et al., 2006). The genes encoding these,
located on chromosomes 4A, 4B, and 4D, are possibly homeoalleles (Ciaffi, M,
Dominici, L, Tanzarella, O and Porceddu, E 1999). Some of the PDILs grouped
together and were conserved in comparable parts; e.g., TaPDIL2-2 and TaPDIL2-1
share 98.5% identity and TaPDIL2-4 and TaPDIL2-5 share 95.7 % identity on cDNA
level; suggesting these genes may be alleles or duplicates.
6.4.2 The PDIL1 group
This study identified six PDIL1 members each in rice and wheat, encoded by five and
six loci, respectively. All PDIL1s present four domains (a, b, b’, and a’) (Freedman et
al., 2002; Wilkinson and Gilbert, 2004), x-linker between b’ and a’, and insertion
/deletion (1-5 aa) in all domains in various rice and wheat members. The length of x-
linker appears conserved except for a two-residue insertion in OsPDIL1-2. The two
213
active site motifs WCGHC (Kemmink et al., 1997) located in domain a at N-terminus
are variant in OsPDIL1-4 (WCAHC) and OsPDIL1-5 (WCERS), and in a’ at C-
terminus in OsPDIL1-5 (WCVDC). Variants are also reported in AtPDIL1-3
(WCGAC) and AtPDIL1-5 and AtPDIL1-6 (WCARS) (Houston et al., 2005). The
thioredoxin (TRX) active site motif WCXXC of PDI shows both disulfide isomerisation
and reduction/oxidation activities (Holmgren, 1985), but the variant WCGHS can
catalyse only disulfide isomerisation and not reduction/oxidation (Woycechowsky et al.,
2000; Serrato et al., 2008). Assays of both activities of these PDIL1s are required to
understand the effects of alterations at the active sites. All PDIL1s in rice and wheat are
predicted to contain the signal peptide at N-terminus for ER targeting (Price et al., 1991)
with varied lengths, from 22 aa (OsPDIL1-2) to 61 aa (TaPDIL1-3). All also show c-
extensions of 24 aa (OsPDIL1-1) to 44 aa (OsPDIL1-4) and the signature C-terminal
tetrapeptide KDEL for ER retention/ retrieval (Freedman et al., 2002), and as expected,
are predicted to be ER-located.
A 60 kDa PDI has been identified in the lumen of ER and found to co-localise with
storage proteins in the dense protein bodies in wheat (Shimoni et al., 1995b). The
mature proteins (TaPDIL1-1, TaPDIL1-2, TaPDIL1-3, TaPDIL1-4) (exclusive of the
SP) are predicted to be 54 kDa, and glycosylation may increase its mass by 6 kDa
(Hirtzlin et al., 1995), making the same (60 kDa) as the reported PDI glycoprotein in
wheat ER (Shimoni et al., 1995b). On amino acid sequence level, TaPDIL1-4 is 85%
identity with TaPDIL1-1, TaPDIl1-2, TaPDIL1-3, and 100% identical to OsPDIL1-1
which regulates segregation of proglutelin and prolamin polypeptides in rice (Takemoto
et al., 2002). This suggests TaPDIL1-4 may be another candidate for storage protein
folding related functions. The orthologues AtPDIL1-1, AtPDIl1-2, AtPDIL1-3, five
PDIL1 in rice and all PDIL1 in wheat may be responsible for ER stress response (Lu
and Christopher, 2008a). The orthologues GmPDIL-1 and GmPDIL-2 play a role in the
folding of storage proteins as both thiol-oxidoreductases and molecular chaperones
(Kamauchi et al., 2007; 2008). Thus all PDIL1s may be responsible for roles in folding
of storage proteins and/or ER stress response, by both enzymatic and chaperone
functions. The possibly allelic genes encoding TaPDIL1-1, TaPDIL1-2, TaPDIL1-3 are
expressed in developing seeds as well as stems and leaves. This is not unexpected, as
the first wheat PDI cDNA (corresponding to TaPDIL1-1, based on its cDNA sequence)
was isolated
214
from root tip library (Shimoni et al., 1995a). TaPDI1-1, TaPDIL1-2, TaPDIL1-3 may
thus play a role in vegetative tissues as well as developing endosperm. Further study
should focus on characterising the genes encoding TaPDIL1-4, TaPDI1-5, TaPDIL1-6
as well as testing for PDIL1 interactions by systems such as the yeast-two hybrid.
6.4.3 The PDIL2 group
This study identified seven PDIL2s (encoded by three loci) in rice, and five in wheat.
Four members in rice and wheat each show N-terminal domains ao and a (Ferrari and
Soling, 1999); OsPDIL2-3, OsPDIL2-3a, OsPDIL2-3b and TaPDIL2-5 present N-
terminal ao and a domains and C-terminal b (Ferrari and Soling, 1999).
Insertions/deletions exists in ao (2 aa), a (2 or 4 aa), b (5-14 aa) in various rice and wheat
members. All PDIL2s shows the conserved active site motif (WCGHC) in ao and a and
all have been predicted to contain a signal peptide at N-terminus for ER targeting (Price
et al., 1991), varying from 18 aa (OsPDIL2-3, OsPDIL2-3a, OsPDIL2-3b) to 29 aa
(OsPDIL2-1, OsPDIL2-1a). OsPDIL2-1, OsPDIL2-1a and TaPDIL2-3 shows part D
(Ferrari and Soling, 1999) with one residue insertion/deletion. OsPDIL2-3 and
TaPDIL2-5 have a c-extension of 34 aa and C-terminal NDEL for ER-retention as
reported in AtFKBP15-2 (He et al., 2004). Further study may be needed to confirm
their cellular locations and confirm the residues essential for ER-retention.
Of the orthologues of OsPDIL2-3 and TaPDIL2-5, human P5 has oxidase, isomerase
and chaperone activities (Ellgaard and Ruddock, 2005; Kim et al., 2009), GmPDIM
plays a role in folding of storage proteins by both thiol-oxidoreductase and chaperone
roles (Kamauchi et al., 2008; 2007), and AtPDIL2-2 and AtPDIL2-3 are responsible for
ER stress response in Arabidopsis (Lu and Christopher, 2008a). These observations
suggest OsPDIL2-3 and TaPDIL2-5 may have similar functions. Further study should
be conducted on these proteins/genes and their expression in developing seeds.
215
6.4.4 The PDIL5 group
Six PDIL5 (encoded by four loci) in rice and 13 in wheat were identified, all presenting
only the a domain (Jeong et al., 2008; Kim et al., 2009) with two main insertions (>6
residues) in OsPDIL5-4, OsPDIL5-4a and TaPDIL5-13. The active sites were variant in
both rice and wheat, i.e., WCKHC (OsPDIL5-1, OsPDIL5-2, TaPDIL5-1, TaPDIL5-2,
TaPDIL5-3, TaPDIL5-4) as found in AtPDIL5-1 or WCYWS (OsPDIL5-4, OsPDIL5-
4a, TaPDIL5-13) in AtPDIL5-3 and AtPDIL5-4 (Houston et al., 2005). As for PDIL1s,
further study should assay the enzymatic activities of these proteins. Five PDIL5
members in rice and twelve in wheat have been predicted to have the SP for N-targeting
(Price et al., 1991), but with varied lengths. OsPDIL5-4a and TaPDIL5-13 show a 90
aa extension at the N-terminus with no predictable cleavage site, suggesting they may
be in locations other than ER. The cytosolic PDIL5-1 in maize is possibly an
orthologue of OsPDIL5-1, TaPDIL5-1, TaPDIL5-2, TaPDIL5-3 and TaPDIL5-4 and
has a molecular chaperone role (Houston et al., 2005). Most members (twelve) contain
extra sequences at the C-terminus, as found in AtPDIL5-2, AtPDIL5-3 and AtPDIL5-4
(Houston et al., 2005). The extension may contain the b domain for peptide binding
(Freedman et al., 2002), but this is as yet unconfirmed. The human ERp18 with N-
terminal SP and C-terminal EDEL is responsible for apoptosis, during ER stress caused
by accumulation of misfolded proteins (Jeong et al., 2008). TaPDIL5-2 shows a similar
structure, including the C-terminal EDEL, suggesting it may function as a chaperone for
regulation of misfolded proteins and requiring further study.
6.4.5 The QSOXL group
This study identified two QSOXL (encoded by one locus) in rice, and one in wheat.
OsQSOXL and OsQSOXLa present a/a’ domain with the motif WCPAC, and an ERV1
domain including its three parts (Thorpe et al., 2002). TaQSOXL lacks the ERV1
domain, and thus may not have the sulfhydryl oxidase activity (Lee et al., 2000; Thorpe
et al., 2002). All QSOXLs have been predicted to have the SP for N-targeting (Price et
al., 1991), with lengths 25 aa or 30 aa. The proteins are predicated to be located in the
ER, but show unusual C-termini, i.e., KNWN and LKKI of OsQSOXL and
OsQSOXLa, compared to those reported for AtQSOX1 and AtQSOX2 (Houston et al.,
2005), needing further study.
216
6.4.6 The APRL group
Seven APRLs (encoded by six loci) in rice and two in wheat were identified. OsAPRL1
and TaAPRL1 exhibit the active sites WCPFC or WCPYC at the C-terminus and lack
the N-terminal active site, as in case of Arabidopsis APRLs (AtAPR1, AtAPR2,
AtAPR3) (Gutierrez-Marcos et al., 1996; Houston et al., 2005). Other members
contained WCPFS at the N-terminus but not at the C-terminus, as in the case of other
Arabidopsis APRLs (AtAPRL4, AtAPRL5, AtAPRL6, AtAPRL7) (Houston et al.,
2005). Further studies should assay for both oxidoreductase and isomerase enzyme
activities of the variant forms. OsAPRL1 and TaAPRL1 shows chloroplast transit
peptides, as noted in PRH-19, PRH-26 and PRH-43 of Arabidopsis (Gutierrez-Marcos
et al., 1996). The intracellular location and biochemical activities of these isoforms also
need further study.
6.4.7 Chromosomal locations of PDILs in rice and wheat
Nineteen loci putatively encoding PDILs in rice are scattered on all chromosomes
except chromosome 10. The putative chromosomal positions of nine genes encoding
wheat PDILs were identified, TaPDIL2-1, TaPDIL2-2, and TaPDIL2-3 being on the
short arms of chromosome 1B or 1D; TaPDIL5-8, TaPDIL5-9, TaPDIL5-11, TaPDIL5-
12 on the long arms of 6A, 6B, and 6D; TaAPRL on 1A, 2A, 2B, and 2D; and TaQsOXL
on the long arm of 1D.
A search for QTLs on these chromosomes shows the total protein content QTL
(AGFU022) (0-116cM on chr6) is located on the 3.6Mb region that may contain the
OsPDIL1-5 gene, and consistency viscosity QTL (CQAG19) (17.3cM on chr6) was
located on the 3.9 Mb region that may contain OsAPRL2 (Appendix IX). QTLs related
to sedimentation value of flour are located on chromosome 6AL, endosperm colour on
2A (Pozniak et al., 2006), mixograph peak time on 1DL (Campbell et al., 2001),
alveograph dough strength on 1A, 1B, and 6B, protein content on 6A (Perretant et al.,
2000), and dough rheological properties on 1AL, 1B, 1D, and 2BL (Ma et al., 2005).
Total protein content and consistency viscosity influence the grain quality traits relevant
to the food industry. Many genes in the area of these QTLs may be potential candidates
for such traits; finer mapping and analysis of any sequence diversity and its effects are
required to address such association.
217
In summary, the identified PDILs in rice and wheat with substituted residues at the
active site motif can be used for confirmation of functional residues required for both
types (oxidoreductase and isomerase) of enzymatic activities of PDI. The functions of
orthologs of TaPDIL1-1, TaPDIL1-2, TaPDIL1-3, TaPDIL1-4, TaPDIL1-5, TaPDIL1-
6, TaPDIL2-5, TaPDIL5-1, TaPDIL5-2, TaPDIL5-3 and TaPDIL5-4 have been
addressed in other organisms, suggesting these may have function in catalytic roles
and/or chaperones in regulation of seed storage protein folding and deposition. Further
studies should focus on characterizing the genes encoding these PDILs and testing the
interaction of various isoforms with each other and other ER chaperones, including
Cyps. All identified TAs encoding PDILs in wheat can be used for further work in gene
characterisation and mapping. There is also a need to look for natural variants of these
genes in wheat cultivars and landraces, as well as wild relatives of wheat of various
ploidy levels, to identify any association with stress response or grain quality.
218
Chapter 7
GENERAL DISCUSSION
219
7 General discussion
A major aim of this thesis was to test the hypothesis that genes encoding cyclophilins
and protein disulphide isomerase-like genes comprise multigene families in wheat, and
to identify new candidate that may be potentially associated with enzymatic and/or
chaperone roles in protein folding, plant development, stress tolerance and/or important
agronomic traits. In order to address this hypothesis, the thesis describes the
identification of such gene families by bioinformatics methods. The experimental
isolation and characterisation of the Cyclophilin B gene in wheat, its physical and
genetic mapping by experimental methods and identification of the corresponding gene
in rice by bioinformatics approaches were other major aims. Identification of the
putative promoter sequences of these genes in wheat (by experimental methods) and in
rice (by bioinformatics tools) and preliminary analysis of expression of selected genes
in wheat are also described. The key results are summarised below, followed by a brief
discussion on future directions
7.1 General discussion
7.1.1 Cyclophilin B shows potential location in the ER
The wheat putative cyclophilin B protein deduced from TaCypB-B in T. aestivum cv
Rosella exhibits a length of 213 amino acids and contains all residues essential for
catalytic activity and immunosuppressant drug binding, as reported for human cytosolic
cyclophiln A and ER-localised cyclophilin B (Pflügl et al. 1993; Price et al., 1991). In
addition, it contains the C-terminal tetrapeptide signal EVPL which has acidic and
hydrophobic residues and partially corresponds to ELPM of purified maize microsomal
cyclophilin (Sheldon and Venis, 1996) and ELPL of Arabidopsis cyclophilins, which
are confirmed to be localized in ER (Saito et al., 1999). The wheat CypB identified
here is thus highly likely to be ER-localized and similar to other such proteins, it is a
strong candidate for diverse enzymatic or chaperone roles in the ER lumen, including
roles in storage protein processes.
7.1.2 Putative ER localized cyclophilin and protein disulphide isomerases may
have roles in ER stress response
In addition to cyclophilin B, two other potential ER localised cyclophilins (TaCYP24-1
and TaCYP26-2) also contain N-terminal ER-targeting sequences and C-terminal
220
extensions (Chapter 5). Orthologues of wheat CypB (TaCYP23-1,Chapter 5) in
Arabidopsis have roles for regulating the protein GNOM required for coordination of
cell polarity along the embryo axis (Grebe et al., 2000) and PP2A (protein phosphatase
2A) for auxin transport and growth response (Jackson and Soll, 1999). TaCYP24-1 has
the same length but not sequence of the C-terminal extension as the human ER-localised
cyclophilin (hCyP-22b/p, NP_000933), acting as a transcriptional inducer (Rycyzyn and
Clevenger, 2002) and playing crucial roles in protecting cells against ER stress (Kim et
al., 2008). Wheat TaCYP24-1 may thus have functions in ER stress response.
The putative promoter sequences of CypB genes in rice and 2n wheat contain relevant
conserved regulatory regulatory sequences such as the GC-rich motif shown to be
critical for initiation of transcription (Pugh, et al., 1991; Sargsyan et al., 2002), the E-
box (Stålberg et al., 1996; Simeone et al., 2006) and Dof core (Vicente-Carbajosa et al.,
1997; Lillemo et al., 2002; Simeone at al., 2006) for tissue specific expression,
endosperm motif (prolamin-box) (Colot et al., 1987; Lillemo et al., 2002; Simeone et
al., 2006) and dyad repeats for endosperm specific expression (Leah et al., 1994;
Lillemo et al., 2002), and AT-rich motif for seed-specific enhancer (Allen et al.,1989;
Marraccini et al., 1999). These support the observed up-regulation of Cyp genes in the
developing wheat endosperm (Grimwade et al., 1996). The presence of an ER stress
responsive element (ERSE-II) (Yoshida et al., 1988; Kokame et al., 2001) in the rice
CypB promoter also supports a role in stress response.
The PDIL1 group in wheat contains the N-terminal signal peptide for ER-targeting and
C-terminal KDEL for ER retention and their orthologues in Arabidopsis may have roles
in ER stress response (Lu and Christopher, 2008a) through folding and regulation of
storage protein by both the thiol-oxidoreductase and chaperone/co-chaperones
(Kamauchi et al., 2007; 2008). The allelic genes encoding TaPDIL1-1, TaPDIL1-2,
TaPDIL1-3 are expressed in developing seeds as well as stems and leaves, the first
cDNA of TaPDIL1-1 having been isolated from root tip library (Shimoni et al., 1995a),
suggesting they may play a role in vegetative tissues as well as developing seeds.
TaPDIL1-4 shows 100% identity to OsPDIL1-1 in rice on amino acid sequence level
and OsPDIL1-1 has been proven to play a key role in regulating the segregation of
proglutelin and prolamin polypeptides during formation of two types of storage protein
bodies in rice grain (Takemoto et al., 2002). TaPDIL2-5 contains N-terminal ER
221
targeting signal peptide, and a C-terminal NDEL, noted in an Arabidopsis ER-PPIase
(AtFKBP15-2) (He et al., 2004), and its orthologues in Arabidopsis (AtPDIL2-2 and
AtPDIL2-3) may have roles for ER stress response (Lu and Christopher, 2008a) through
folding and regulation of storage proteins by its roles as a thiol-oxidoreductase and
chaperone (Kamauchi et al., 2008; 2007). TaPDIL5-2 shows a similar structure to
human ERp18, which is responsible for apoptosis during ER stress caused by
accumulation of misfolded proteins (Jeong et al., 2008); a similar role for TaPDIL5-2 is
thus plausible and needs to be investigated.
7.1.3 Plant cyclophilins in other cellular locations show a variety of functions
The Cyps show a range of functions through both catalytic and chaperone roles in
different cellular locations. Orthologues of cytosolic Cyps (TaCYP18-1, TaCYP18-2,
TaCYP19) have roles in regulating plant growth in tomato (Oh et al., 2006) and in
Solanum sogarandinum (Kielbowicz-Matuk et al., 2007), and are involved in stress
response in potato induced by pathogens (Dubery, 2007). Its orthologue in Arabidopsis
is required for vegetative phase change (Berardini et al., 2001) by regulation of
miR156-regulated members (Smith et al., 2009). An orthologue of the chloroplastic
TaCYP26-1 functions in repairing the photodamaged photosystem II (PSII) in
Arabidopsis (Cai et al., 2008). TaCYP46-2 contains a leucine zipper motif at N-
terminus and its potential orthologue AtCYP38 - with conserved functional residues and
a leucine zipper motif - plays a critical role in the assembly and maintenance of PSII
supercomplexes (Fu et al., 2007; Sirpio et al., 2008). Similar roles seem plausible for
the relevant wheat and rice orthologues.
7.2 Future directions
The findings of the current study have provided invaluable information about the
cyclophilin B genes in wheat. Further, analysis of the Cyp and PDI superfamilies has
led to identification of additional ER-localised isoforms of these foldase enzymes, and
several members which have orthologues in other species known to play chaperone or
regulatory roles. The work has thus led to identification of several new candidates for
potential roles in folding and regulation of storage protein processes and ER stress
response. The following research directions should produce further insights into these
areas:
222
• Data on wheat orthologues at rice CypB locus indicates that wheat CypB gene
(designated as TaCypB7A, TaCypB7B, TaCypB7D in chapter 4; TaCYPB23-1 in
Chapter 5) may be on chromosomes 1 and 7. The location on chromosome 7 has
been confirmed (chapter 4) but that on chromosome 1 is currently unsupported and
needs further work, e.g., by Southern blotting of DNA of various ploidy levels, or
use of other stocks of nullisomic/tetrasomic or deletion lines.
• The putative wheat CypB with unusual C-terminal EVPL is predicted to be located
in ER. While varying from the classical KDEL, it is partially similar to variant
sequences of some ER-Cyps in maize and Arabidopsis. This CypB is thus a strong
candidate for ER-localisation and consequent roles in storage protein processes. Its
cellular location needs to be confirmed, e.g., by immunological techniques and
manipulation of gene sequence.
• Its roles can be investigated by expression of the protein, and/or study of deletion or
other mutant lines, and/or studies of its orthologues in rice or Arabidopsis.
• Several other identified putative ER-localised wheat Cyps may (also) play important
roles as catalysts/chaperones in storage protein folding and protein body formation.
• The identified TA sequences encoding ER-localised wheat Cyps provide important
tools for full characterisation of their genes in T. aestivum and/or its progenitors as
well as identifying their functions. The cellular locations of TaCYP24-1 and
TaCYP26-2 also need to be confirmed and their ER retention addressed.
• ER-Cyps, and Cyps with leucine zippers, may form homo- or heterodimers or
cooperate with other chaperones in performing their roles. The protein-protein
interactions within/between Cyps and/or with other ER chaperones and/or storage
proteins can be tested by systems such as yeast-two hybrid.
• All identified wheat/rice Cyps with substitutions at catalytic sites can be tested for
drug binding, enzymatic and/or chaperone abilities for confirmation of essential
residues.
• The putative ER-localised PDILs (TaPDIL1-4, TaPDIL1-5, TaPDIL1-6, TaPDIL2-
5, TaPDIL5-2) in wheat may similarly play important roles in catalysis/regulation of
storage protein folding and deposition, and/or response to internal ER stress that
occurs due to intense protein synthetic activities. The identified TAs provide
important tools for characterisation of their genes and functions in T. aestivum
and/or its progenitors.
223
• Of these, TaPDIL1-4 is 100% identical to OsPDIL1-1 on amino acid level and may
be duplicates of TaPDIL1-1, TaPDIL1-2, and TaPDIL1-3 with 83-84% identity on
coding sequence level and 85% identity on amino acid level. OsPDIL1-1 is proven
to regulate segregation of proglutelin and prolamin polypeptides in rice (Takemoto
et al., 2002). The molecular weight of glycosylation protein TaPDIL1-4 is 60 kDa,
close to the reported PDI glycoprotein co-localising with storage proteins in the
dense protein bodies in wheat endosperm (Shimoni et al., 1995). TaPDIL1-4 may
thus be another candidate for storage protein folding and assortment roles, requiring
detailed study including gene characterisation and mapping.
• All identified Cyp and PDIL genes can be used for mapping purposes and
identification of any QTL-related wheat quality traits co-localising with these genes.
224
References
ABARE (2008a) Australia Bureau of Agriculture and Resource Economics, Australia
crop report 2008
(http://www.abareconomics.com/publications_html/cr/cr_08/cr08_Dec.pdf;
accessed 03 2009).
ABARE (2008b) Australia Bureau of Agriculture and Resource Economics, Australia
commodity 2006
(http://www.abareconomics.com/publications_html/ac/ac_06/ac_06.html;
accessed
03 2009).
Aguilar V, Stamp P, Winzeler M, Winzeler H, Schachermayr G, Keller B, Zanetti S,
Messmer MM (2005) Inheritance of field resistance to Stagonospora nodorum
leaf and glume blotch and correlations with other morphological traits in
hexaploid wheat (Triticum aestivum L.). Theor Appl Genet 111: 325-336.
Alergand T, Peled-Zehavi H, Katz Y, Danon A (2006) The chloroplast protein disulfide
isomerase RB60 reacts with a regulatory disulfide of the RNA-binding protein
RB47. Plant Cell Physiol 47: 540-548.
Allaby R, Brown T (2000) Identification of a 5S rDNA spacer type specific Triticum
urartu and wheats containing the T. urartu genome. Genome 43: 250-254.
Allen RD, Bernier F, Lessard PA, Beachy RN (1989) Nuclear Factors Interact with a
Soybean [beta]-Conglycinin Enhancer. Plant Cell 1: 623-631.
Altschuler Y, Galili G (1994) Role of conserved cysteines of a wheat gliadin in its
transport and assembly into protein bodies in Xenopus oocytes. J Biol Chem
269: 6677–6682.
Altschuler Y, Harel R, Galili G (1993) Role of the N- and C-terminal regions of a wheat
-gliadin in its transport via the endoplasmic reticulum of Xenopus oocytes.
Plant Cell 5: 443–450.
Anderson S, Gallinger S, Roder J, Frey J, Young H, Ortaldo J (1993) A cyclophilin-
related protein involved in the function of natural killer cells. Proc Natl Acad Sci
U S A. 90: 542–546.
Appella E, Anderson C (2007) Post-translational modifications – S-palmitoylation,
proline isomerization, disulfide bond isomerization. FEBS J 274: 5201-5201.
225
Appenzeller-Herzog C, Ellgaard L (2008a) The human PDI family: Versatility packed
into a single fold. Biochim Biophys Acta 1738: 535-548.
Appenzeller-Herzog C, Ellgaard L (2008b) In vivo reduction-oxidation state of protein
disulfide isomerase: The two active sites independently occur in the reduced and
oxidized forms. Antioxid Redox Signal 10: 55-64.
Arcalis E, Marcel S, Altmann F, Kolarich D, Drakakaki G, Fischer R, Christou P,
Stoger E (2004) Unexpected deposition patterns of recombinant proteins in post-
endoplasmic reticulum compartments of wheat endosperm. Plant Physiol. 136:
3457-3466.
Batey IL, Hayden MJ, Cai S, Sharp PJ, Cornish GB, Morell MK, Appels R (2002)
Genetic mapping of commercially significant starch characteristics in wheat
crosses. Aust J Agric Res 52: 1287-1296.
Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill
EW, Sayen MR, Gottlieb RA, Dorn II GW, Bobbins J, Molkentin JD (2005)
Loss of cyclophilin D reveals a critical role for mitochondrial permeability
transition in cell death. Nature 434: 658-662.
Barisic K MS, Noegel A. A, Gerisch G, Segall JE (1991) cDNA sequence of cyclophilin
from Dictyostelium discoideum. Dev Genet. 12: 50-53.
Barro F, Rooke L, Bekes F, Gras P, Tatham A, Fido R, Lazzeri P, Shewry P, Barcelo P
(1997) Transformation of wheat with high molecular weight subunit genes
results in improved functional properties. Nat Biotechnol 15: 1295 - 1299.
Baynton CE, Potthoff SJ, McCullough AJ, Schuler MA (1996) U-rich tracts enhance 3′
splice site recognition in plant nuclei. Plant J 10: 703-711.
Berardini T, Bollman K, Sun H, PR S (2001) Regulation of vegetative phase change in
Arabidopsis thaliana by cyclophilin 40. Science 291: 2405-2407
Birney E, Kumar S, Krainer A (1993) Analysis of the RNA-recognition motif and RS
and RGG domains: conservation in metazoan pre-mRNA splicing factors
Nucleic Acids Res 21: 5803-5816.
Blanco A, Simeone R, Gadaleta A (2006) Detection of QTLs for grain protein content
in durum wheat. Theor Appl Genet 112: 1195–1204.
Boden G, Duan X, Homko C, Molina E, Song W, Perez O, Cheung P, Merali S (2008)
Increase in endoplasmic reticulum stress–related proteins and genes in adipose
tissue of obese, insulin-resistant individuals. Diabetes 57: 2438-2444.
226
Boldbaatar D, Kilonzo R, Battur B, Umemiya R, Liao M, Tanaka T, Xuan X & Fujisaki
K (2008) Identification of two forms of cyclophilin from the hard tick
Haemaphysalis longicornis. Process Biochem. 43: 615-625
Bose S, Mucke M, Freedman RB (1994) The characterization of a cyclophilin-type
peptidyl prolyl cis-trans-isomerase from the endoplasmic-reticulum lumen.
Biochem J. 300: 871-875.
Bossard MJ, Koser PL, Brandt M, Bergsma DJ, Levy MA (1991) A single Trp121 to
Ala121 mutation in human cyclophilin alters cyclosporin A affinity and
peptidyl-prolyl isomerase activity. Biochem Biophys Res Commun 176: 1142-
1148.
Boston RS, Viitanen PV, Vierling E (1996) Molecular chaperones and protein folding
in plants. Plant Molecular Biology 32: 191 - 222.
Bozkurt O, Unver T, Akkaya M (2008) Genes associated with resistance to wheat
yellow rust disease identified by differential display analysis. Physiol Mol Plant
Path 71: 251-259.
Brazin KN, Mallis RJ, Fulton DB, Andreotti AH (2002) Regulation of the tyrosine
kinase Itk by the peptidyl-prolyl isomerase cyclophilin A. Proc Natl Acad Sci
USA 99: 1899–1904.
Breiman A, Graur D (1995) Wheat Evolution. Israel Journal of Plant Sciences 43: 85-
89.
Buchholz WG, Harris-Haller L, DeRose RT, Hall TC (1994) Cyclophilins are encoded
by a small gene family in rice. Plant Mol Biol 25: 837 - 843.
Bulleid N & Freedman R (1988) Defective co-translational formation of disulphide
bonds in protein disulphide-isomerase-deficient microsomes. Nature 335: 649 -
651.
Cai H, Wang C, Tsou C (1994) Chaperone-like activity of protein disulfide isomerase in
the refolding of a protein with no disulfide bonds. J Biol Chem 269: 24550-
24552.
Cai W, Ma J, Guo J, Zhang L (2008) Function of ROC4 in the efficient repair of
photodamaged photosystem II in Arabidopsis. Photochem Photobiol 84: 1343-
1348
Campbell K, Finney P, Bergman C, Gualberto D, Anderson J, Giroux M, Siritunga D,
Zhu J, Gendre F, Roué C, Vérel A, Sorrells M (2001) Quantitative trait loci
227
associated with milling and baking quality in a soft x hard wheat cross Crop Sci
41: 1275-1285
Carmichael D, Morin J, Dixon J (1977) Purification and characterization of a
thiol:protein disulfide oxidoreductase from bovine liver. J Biol Chem 252: 7163-
7167.
Chalmers KJ, Campbell AW, Kretschmer J, Karakousis A, Henschke PH, Pierens S,
Harker N, Pallotta M, Cornish GB, Sharifiou MR, Rampling LR, McLauchlan
A, Daggard G, Sharp PJ, Holton TA, Sutherland MW, Appels R, Langridge P
(2001) Construction of three linkage maps in bread wheat, Triticum aestivum.
Aust J Agric Res 52: 1089-1119.
Chen AP, Wang GL, Qu ZL, Lu CX, Liu N, Wang F, Xia GX (2007) Ectopic
expression of ThCYP1, a stress-responsive cyclophilin gene from Thellungiella
halophila, confers salt tolerance in fission yeast and tobacco cells. Plant Cell
Rep 26: 237-245.
Cho E, Kim M (2008) A red algal cyclophilin has an effect on development and growth
in Nicotiana tabacum. Plant Physiol Biochem 46: 868-874
Chou I, Gasser C (1997) Characterization of the cyclophilin gene family of Arabidopsis
thaliana and phylogenetic analysis of known cyclophilin proteins. Plant Mol
Biol 35: 873 - 892.
Ciaffi M, Paolacci A, D'Aloisio E, Tanzarella O, Porceddu E (2006) Cloning and
characterization of wheat PDI (protein disulfide isomerase) homoeologous genes
and promoter sequences. Gene 366: 209–218.
Ciaffi M, Dominici L, Tanzarella O, Porceddu E (1999) Chromosomal assignment of
gene sequences coding for protein disulphide isomerase (PDI) in wheat. Theor
Appl Genet 98: 405 - 410.
Ciaffi M, Tozzi L, Borghi B, Corbellini M & Lafiandra D (1996) Effect of heat shock
during grain filling on the gluten protein composition of bread wheat. J Cereal
Sci 24: 91-100
Colot V, Robert LS, Kavanagh TA, Bevan MW, Thompson RD (1987) Localization of
sequences in wheat endosperm protein genes which confer tissue-specific
expression in tobacco. EMBO J 6: 3559-3564.
Coughlan J, Hastings C, Winfrey R (1996) Molecular characterisation of plant
endoplasmic reticulum identification of protein disulfide-isomerase as the major
reticuloplasmin. Eur J Biochem 235: 215 - 224.
228
Creighton T, Hillson D, Freedman R (1980) Catalysis by protein-disulphide isomerase
of the unfolding and refolding of proteins with disulphide bonds. J Mol Biol
142: 43-62.
Crofts AJ, Washida H, Okita TW, Ogawa M, Toshihiro K, Satoh H (2004) Targeting of
proteins to endoplasmic reticulum-derived compartments in plants. The
importance of RNA localization. Plant Physiol 136: 3414-3419
Crooke E & Wickner W (1987) Trigger factor: a soluble protein that folds pro-OmpA
into a membrane-assembly-competent form. Proc Natl Acad Sci USA 84: 5216–
5220.
Darby N, Creighton T (1995) Functional properties of the individual thioredoxin-like
domains of protein disulfide isomerase. Biochemistry 34: 11725-11735.
Darby N, Penkaa E, Vincentelli R (1998) The multi-domain structure of protein
disulfide isomerase is essential for high catalytic efficiency. J Mol Biol 276:
239-247
Denecke J, De Rycke R, Botterman J (1992) Plant and mammalian sorting signals for
protein retention in the endoplasmic reticulum contain a conserved epitope.
EMBO J 11: 2345-2355.
Derkx P, Madrid S (2001) The foldase CYPB is a component of the secretory pathway
of Aspergillus niger and contains the endoplasmic reticulum retention signal
HEEL. Mol Genet Genom 266: 537-545.
Digeon JF, Guiderdoni E, Alary R, Nicole MF, Joudrier P, Gautier MF, (1999) Cloning
of a wheat puroindoline gene promoter by IPCR and analysis of promoter
regions required for tissue-specific expression in transgenic rice seeds. Plant
Mol Biol 39: 1101-1112.
Dixon D, Van Lith M, Edwards R, Benham A (2003) Cloning and initial
characterization of the Arabidopsis thaliana endoplasmic reticulum
oxidoreductins. Antioxid Redox Signal 5: 389-396.
Dominguez-Solis J, He Z, Lima A, Ting J, Buchanan B, Luan S (2008) A cyclophilin
links redox and light signals to cysteine biosynthesis and stress responses in
chloroplasts. Proc Nat Acad Sci USA 105: 16386-16391.
Dubery I (2007) An elicitor- and pathogen-induced cDNA from potato encodes a stress-
responsive cyclophilin. Biol Plant 51: 327-332.
229
Dubcovsky J, Dvorak J (2007) Genome plasticity a key factor in the success of
polyploid wheat under domestication. Science 316: 1862 - 1866.
Dubourg B, Kamphausen T, Weiwad M, Jahreis G, Feunteun J, Fischer G, Modjtahedi
N (2004) The human nuclear SRcyp is a cell cycle-regulated cyclophilin. J Bio
Chem 279: 22322-22330.
Duina A, Chang H, Marsh J, Lindquist S, Gaber R (1996) A cyclophilin function in
hsp90-dependent signal transduction. Science 274: 1713-1715.
Dupont F, Altenbach S (2003) Molecular and biochemical impacts of environmental
factors on wheat grain development and protein synthesis J Cereal Sci 38: 133-
146
DuPont F, Hurkman W, Vensel W, Chan R, Lopez R, Tanaka C, Altenbach S (2006)
Differential accumulation of sulfur-rich and sulfur-poor wheat flour proteins is
affected by temperature and mineral nutrition during grain development. J
Cereal Sci 44: 101-112.
DuPont F, William J, Charlene K, Ronald C (1998) BiP, HSP70, NDK and PDI in
wheat endosperm. I. Accumulation of mRNA and protein during grain
development. Physiol Plantarum 103: 70-79.
Dvorak J, Luo MC, Yang ZL, Zhang HB (1998) The structure of the Aegilops tauschii
gene pool and the evolution of hexaploid wheat. Theor Appl Genet 97: 657-670.
Edvardsson A, Eshaghi S, Vener A, Andersson B (2003) The major peptidyl-prolyl
isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel
cyclophilin TLP20. FEBS Lett 542: 137-141.
Ellgaard L, Ruddock L (2005) The human protein disulphide isomerase family:
substrate interactions and functional properties. EMBO Rep 6: 28-32.
Endo TR, Gill BS (1996) The deletion stocks of common wheat. J Hered 87: 295-307.
Farquharson K (2008) A protein disulfide isomerase plays a role in programmed cell
death. Plant cell : DOI: 10.1105/tpc.108.200810.
Ferrari D, Soling H (1999) The protein disulphide-isomerase family: unravelling a
string of folds. Biochem J 339: 1-10.
Feuillet C, Langridge P, Waugh R (2008) Cereal breeding takes a walk on the wild side.
Trends Genet 24: 24-32.
230
Fischer G, Bang H, Mech C (1984) Determination of enzymatic catalysis for the cis-
trans-isomerization of peptide binding in proline-containing peptides. Biochim
Biophys Acta 43: 1101-1111.
Fonseca C, Soiffer R, Ho V, Vanneman M, Jinushi M, Ritz J, Neuberg D, Stone R,
DeAngelo D, Dranoff G (2009) Protein disulfide isomerases are antibody targets
during immune-mediated tumor destruction. Blood 113: 1681-1688.
Forster M, Sivick K, Park Y, Arvan P, Lencer WI, Tsai B (2006) Protein disulfide
isomerase-like proteins play opposing roles during retrotranslocation. J Cell Biol
173: 853–859.
Freedman R, Klappa P, Ruddock L (2002) Protein disulfide isomerases exploit synergy
between catalytic and specific binding domains. EMBO Rep 3: 136-140.
Frigerio G, Pelham HRB (1993) A Saccharomyces cerevisiae cyclophilin resident in the
endoplasmic reticulum. J Mol Biol 233: 183-188
Fu A, He Z, Hye S, Lima A, Buchanan B, Luan S (2007) A chloroplast cyclophilin
functions in the assembly and maintenance of photosystem II in Arabidopsis
thaliana. Proc Natl Acad Sci USA 104: 15947-15952
Fu X (1995) The superfamily of arginine/serine-rich splicing factors. RNA 1: 663-680.
Fu X, Wang P, Zhu B (2008) Protein disulfide isomerase is a multifunctional regulator
of estrogenic status in target cells. J Steroid Biochem Mol Biol 112: 127-137
Fulgosi H, Vener A, Altschmied L, Herrmann R, Andersson B (1998) A novel multi-
functional chloroplast protein: identification of a 40 kDa immunophilin-like
protein located in the thylakoid lumen. EMBO J 17: 1577-1587.
Galat A (1993) Peptidylproline cis-trans-isomerases: immunophilins. Eur J Biochem
216: 689-707.
Galat A (1999) Variations of sequences and amino acid compositions of proteins that
sustain their biological functions: an analysis of the cyclophilin family of
proteins. Arch Biochem Biophys 371: 149-162.
Galat A (2003) Peptidylprolyl cis/trans isomerases (immunophilins): biological
diversity targets functions. Curr Top Med Chem 3: 1315-1347.
Galat A (2004) Function-dependent clustering of orthologues and paralogues of
cyclophilins. Proteins Struct Funct Bioinf 56: 808-820.
Galili G (2004) ER-derived compartments are formed by highly regulated processes and
have special functions in plants Plant Physiol 136: 3411-3413.
231
Galili G, Sengupta-Gopalan C, Ceriotti A (1998) The endoplasmic reticulum of plant
cells and its role in protein maturation and biogenesis of oil bodies. Plant Mol
Biol 38: 1-29.
Galili S AY, Millet E, Feldman M. (2000 ) RFLP-based analysis of three RbcS
subfamilies in diploid and polyploid species of wheat. Mol Gen Genet. 263:
674-680.
Gama A, Santos D, Lopes da Silva JA (2000) Influence of wheat polysaccharides on the
rheological properties of gluten and doughs. In: Shewry P, Tatham AS (eds),
Wheat gluten. The Royal Society of Chemistry, Cambridge, pp. 503-506.
Gasser B, Sauer M, Maurer M, Stadlmayr G, Mattanovich D (2007) Transcriptomics-
based identification of novel factors enhancing heterologous protein secretion in
yeasts. Appl Environ Microbiol 73: 6499-6507.
Gasser CS, Gunning DA, Budelier KA, Brown SM (1990) Structure and expression of
cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and
production of active tomato cyclophilin in Escherichia coli. Proc Natl Acad Sci
USA 87: 9519-9523.
Gavela Y, von Heijne G (1990) A conserved cleavage-site motif in chloroplast transit
peptides. FEBS Lett 261: 455-458.
Gianibelli MC, Larroque OR, MacRitchie F, Wrigley CW (2001) Biochemical, genetic,
and molecular characterization of wheat endosperm proteins. AACC 78: 635-
646.
Gill B, Kimber G (1974) Giemsa C-banding and the evolution of wheat. Proc Nat Acad
Sci USA 71: 4086-4090.
Godoy A, Lazzaro A, Casalongué C, Segundo SB (2000) Expression of a Solanum
tuberosum cyclophilin gene is regulated by fungal infection and abiotic stress
conditions. Plant Sci 152: 123-134
Goldberger R, Epstein C, Anfinsen C (1964) Purification and properties of a
microsomal enzyme system catalyzing the reactivation of reduced ribonuclease
and lysozyme. J Biol Chem 239: 1406-1410.
Grebe M, Gadea J, Steinmann T, Kientz M, Rahfeld JU, Salchert K, Koncz C, Jürgens
G (2000) A conserved domain of the Arabidopsis GNOM protein mediates
subunit interaction and Cyclophilin 5 binding. Plant Cell 12: 343-356.
232
Grimwade B, Tatham AS, Freedman RB, Shewry PR, Napier JA (1996) Comparison of
the expression patterns of genes coding for wheat gluten proteins and proteins
involved in the secretory pathway in developing caryopses of wheat. Plant Mol
Biol 30: 1067-1073.
Groos C, Bervas E, Chanliaud E, Charmet G (2007) Genetic analysis of bread-making
quality scores in bread wheat using a recombinant inbred line population. Theor
Appl Genet 115: 313-323.
Gruber C, Cemazar M, Clark R, Horibe T, Renda R, Anderson M, Craik D (2007) A
Novel plant protein-disulfide isomerase involved in the oxidative folding of
cysteine knot defense proteins. J Biol Chem 282: 20435-20446.
Gruber C, Cemazar M, Heras B, Martin J, Craik D (2006) Protein disulfide isomerase:
the structure of oxidative folding. Trends Biochem Sci 31: 455-464
Gruber C, Cemazar M, Mechler A, Martin L, Craik D (2009) Biochemical and
biophysical characterization of a novel plant protein disulfide isomerase.
Biopolymers 92: 35-43.
Grynberg A, Nicolas J, Drapron R (1977) Presence of a protein disulfide isomerase (EC
5.3.4.1) in wheat germ. C R Acad Sci Hebd Seances Acad Sci D 284: 235-238.
Gullerova M, Barta A, Lorkovic Z (2007) Rct1, a nuclear RNA recognition motif-
containing cyclophilin, regulates phosphorylation of the RNA polymerase II C-
terminal domain. Mol Cell Biol 27: 3601-3611
Gullerova M, Barta A, Lorkovi ZJ (2006) AtCyp59 is a multidomain cyclophilin from
Arabidopsis thaliana that interacts with SR proteins and the C-terminal domain
of the RNA polymerase II RNA 12: 631-643.
Gutierrez-Marcos J, Roberts M, Campbell E, Wray J (1996) Three members of a novel
small gene-family from Arabidopsis thaliana able to complement functionally an
Escherichia coli mutant defective in PAPS reductase activity encode proteins
with a thioredoxin-like domain and “APS reductase” activity Proc Nat Acad Sci
USA 93: 13377-13382.
Haendler B, Keller R, Hiestand P, Kocher H, Wegmann G, Movva N (1989) Yeast
cyclophilin: isolation and characterization of the protein, cDNA and gene. Gene
83: 39-46.
Hamelberg D, Mecammon J (2009) Mechanistic insight into the role of transition-state
stabilization in cyclophilin A. J Am Chem Soc 131: 147-152
233
Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW (1984)
Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226:
544-547.
Hara-Nishimura I, Matsushima R, Shimada T, Nishimura M (2004) Diversity and
formation of endoplasmic reticulum-derived compartments in plants. Are these
compartments specific to plant cells? Plant Physiol 136: 3435-3439
Harding M, Galat A, Uehling D, Schreiber S (1989) A receptor for the immuno-
suppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 341: 758-
760.
Harding M, Handschumacher R, Speicher D (1986) Isolation and amino acid sequence
of cyclophilin. J Biol Chem 261: 8547-8555.
Hatahet F, Ruddock L (2007) Substrate recognition by the protein disulfide isomerases.
FEBS J 274: 5223-5234.
Hayano T, Kikuchi M (1995) Cloning and sequencing of the cDNA encoding human
P5. Gene 164: 377-378.
Hayano T, Kikuchi M (1995) Molecular cloning of the cDNA encoding a novel protein
disulfide isomerase-related protein (PDIR). FEBS Lett 372: 210-214
He P, Friebe BR, Gill BS, Zhou JM (2003) Allopolyploidy alters gene expression in the
highly stable hexaploid wheat. Plant Mol Biol 52: 401-414.
He Z, Li L, Luan S (2004) Immunophilins and parvulins. Superfamily of peptidyl prolyl
isomerases in Arabidopsis. Plant Physiol. 134: 1248-1267.
Heck JA, Meng X, Frick DN (2009) Cyclophilin B stimulates RNA synthesis by the
HCV RNA dependent RNA polymerase. Biochem Pharmacol 77: 1173-1180.
Herman E, Larkins B (1999) Protein storage bodies and vacuoles. Plant Cell 11: 601-
614.
Herman E, Schmidt M (2004) Endoplasmic reticulum to vacuole trafficking of
endoplasmic reticulum bodies provides an alternate pathway for protein transfer
to the vacuole. Plant Physiol 136: 3440-3446.
Herrler M, Bang H, Marahiel M (1994) Cloning and characterization of ppiB, a Bacillus
subtilis gene which encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans
isomerase. Mol Microbiol 11: 1073-1083.
234
Hirtzlin J, Färber PM, Franklin RM, Bell A (1995) Molecular and biochemical
characterization of a Plasmodium falciparum cyclophilin containing a cleavable
signal sequence. FEBS J 232: 765-772.
Holmgren A (1985) Thioredoxin. Ann Rev Biochem 54: 237-271.
Horibe T, Kikuchi M, Kawakami K (2008) Interaction of human protein disulfide
isomerase and human P5 with drug compounds: Analysis using biosensor
technology. Process Biochem 43: 1330-1337.
Hornbogen T, Pieper R, Hoffmann K, Kleinkauf H, Zocher R (1992) Two new
cyclophilins from Fusarium sambucinum and Aspergillus niger: resistance of
cyclophilin/cyclosporin A complexes against proteolysis. Biochem Biophys Res
Commun 187: 791-796.
Houston N, Fan C, Xiang Q, Schulze J (2005) Phylogenetic analyses identify 10 classes
of the protein disulfide isomerase family in plants, including single-domain
protein disulfide isomerase-related proteins. Plant Physiol 137: 762-778.
Huang S, Sirikhachornkit A, Su X, Faris J, Gill B, Haselkorn R, Gornicki P (2002)
Genes encoding plastid acetyl-CoA carboxylase and 3-phosphoglycerate kinase
of the Triticum/Aegilops complex and the evolutionary history of polyploid
wheat. Proc Natl Acad Sci USA 99: 8133-8138.
Huang D, Chen H, Lin Y (2005) Isolation and expression of protein disulfide isomerase
cDNA from sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57') storage roots.
Plant Sci 169: 776-784.
Huang XQ, Cloutier S, Lycar L, Radovanovic N, Humphreys DG, Noll JS, Somers DJ,
Brown PD (2006) Molecular detection of QTLs for agronomic and quality traits
in a doubled haploid population derived from two Canadian wheats (Triticum
aestivum L.) Theor Appl Genet 113: 753-766.
Hur S, Bruice T (2002) The mechanism of cis-trans isomerization of prolyl peptides by
cyclophilin. J Am Chem Soc 124: 7303-7313.
Hurkman W, Vensel W, Tanaka C, Whitehand L, Altenbach S (2009) Effect of high
temperature on albumin and globulin accumulation in the endosperm proteome
of the developing wheat grainq. J Cereal Sci 49: 12-23.
Hwang I (2008) Sorting and anterograde trafficking at the Golgi apparatus. Plant
Physiol 148: 673-683
235
Jackson KS, Soll D (1999) Mutations in a new Arabidopsis cyclophilin disrupt its
interaction with protein phosphatase 2A. Mol Gen Genet 262: 830-838.
Jacquot J, Gelhaye E, Rouhier N, Corbier C, Didierjean C, Aubry A (2002)
Thioredoxins and related proteins in photosynthetic organisms: molecular basis
for thiol dependent regulation. Biochem Pharmacol 64: 1065-1069.
Jeong W, Lee D, Park S, Rhee S (2008) ERp16, an endoplasmic reticulum-resident
thiol-disulfide oxidoreductase. J Biol Chem 283: 25557-25566.
Jia G, Chen P, Qin G, Bai G, Wang X, Wang S, Zhou B, Zhang S, Liu D (2005) QTLs
for Fusarium head blight response in a wheat DH population of
Wangshuibai/Alondra‘s’. Euphytica 146: 183-19.
Jiang J, Gill B (1994) New 18S·26S ribosomal RNA gene loci: chromosomal landmarks
for the evolution of polyploid wheats. Chromosoma 103: 179-185.
Johnson JC, Clark B, Bhave M (2001) Isolation and characterisation of cDNAs
encoding protein disulphide isomerases and cyclophilins in wheat. J Cereal Sci
34: 159-171.
Johnson JC, Bhave M (2004a) Molecular characterisation of the protein disulphide
isomerase genes of wheat. Plant Sci 167: 397-410.
Johnson JC, Bhave M (2004b) Characterisation and physical mapping of cyclophilin A
genes and identification of new classes of cyclophilins in wheat. J Cereal Sci 40:
137-150.
Johnson JC, Appels R, Bhave M (2006) The PDI genes of wheat and their syntenic
relationship to the esp2 locus of rice. Funct Integr Genomics 6: 104-121.
Joseph JD, Heitman J, Means AR (1999) Molecular cloning and characterization of
Aspergillus nidulans cyclophilin B. Fungal Genet Biol 27: 55-66.
Kainuma K, Ookura T, Kawamura Y (1995) Purification and characterization of protein
disulfide isomerase from soybean. J Biochem 117: 208-215.
Kallen J, Claus S, Mauro G, Gerhard W, Hans W, Kurt W, Malcolm D (1991) Structure
of human cyclophilin and its binding site for cyclosporin A determined by X-ray
crystallography and NMR spectroscopy. Nature 353: 276 - 279.
Kallen J, Walkinshaw MD (1992) The X-ray structure of a tetrapeptide bound to the
active site of human cyclophilin A. FEBS Lett 300: 286-290.
236
Kamauchi S, Wadahama H, Iwasaki K, Nakamoto Y, Nishizawa K, Ishimoto M,
Kawada T, Urade R (2008) Molecular cloning and characterization of two
soybean protein disulfide isomerases as molecular chaperones for seed storage
proteins. FEBS J 275: 2644-2658.
Kammholz J, Campbell AW, Sutherland MW, Hollamby GJ, Martin PJ, Eastwood RF,
Barclay I, Wilson RE, Brennan PS, Sheppard JA (2001) Establishment and
characterisation of wheat genetic mapping populations. Aust J Agr Res 52: 1079
- 1088.
Kawagoe Y, Suzuki K, Tasaki M, Yasuda H, Akagi K, Katoh E, Nishizawa N, Ogawa
M, Takaiwa F (2005) The critical role of disulfide bond formation in protein
sorting in the endosperm of rice. Plant Cell 17: 1141-1153.
Ke H, Zydowsky LD, Liu J, Walsh CT (1991) Crystal structure of recombinant human
T-cell cyclophilin A at 2.5 Å resolution. Proc Natl Acad Sci USA 88: 9483-
9487.
Keller M, Keller B, Schachermayr G, Winzeler M, Schmid JE, Stamp P, Messmer MM
(1999) Quantitative trait loci for resistance against powdery mildew in a
segregating wheat x spelt population. Theor Appl Genet 98: 903-912.
Kemmink J, Darby N, Dijkstra K, Nilges M, Creighton T (1997) The folding catalyst
protein disulfide isomerase is constructed of active and inactive thioredoxin
modules. Curr Biol 7: 239-245.
Kiełbowicz-Matuk A, Rey P, Rorat T (2007) The abundance of a single domain
cyclophilin in Solanaceae is regulated as a function of organ type and high
temperature and not by other environmental constraints. Physiol Plant 131: 387-
398.
Kim J, Choi T, Ding Y, Kim Y, Ha K, Lee K, Kang I, Ha J, Kaufman R, Lee J, Choe
W, Kim S (2008) Overexpressed cyclophilin B suppresses apoptosis associated
with ROS and Ca2+ homeostasis after ER stress. J Cell Sci 121: 3636-3648.
Kim J, Lee M, Jang C, Heo H, Park K, Kim H, Kang M, Kim S, Kim J, Hyun D, Seo Y
(2008) Molecular characterization of a cDNA encoding disulfide isomerase
during barley kernel development. Russ J Plant Physiol 55: 676-686.
Kim WT, Vincent RF, Hari BK, Thomas WO (1988) Formation of wheat protein
bodies: Involvement of the Golgi apparatus in gliadin transport. Planta 176: 173-
182.
237
Kim Y, Kang K, Kim I, Lee Y, Oh C, Ryoo J, Jeong E, Ahn K (2009) Molecular
mechanisms of MHC class I-antigen processing: redox considerations. Antioxid
Redox Signal 11: 907-936.
Kimber G, Athwal R (1972) A reassessment of the course of evolution of wheat. Proc
Natl Acad Sci USA 69: 912–915.
Klappa P, Freedman RB, Zimmermann R (1995) Protein disulphide isomerase and a
lumenal cyclophilin-type peptidyl prolyl cis-trans isomerase are in transient
contact with secretory proteins during late stages of translocation. Eur J
Biochem 232: 755-764.
Klappa P, Freedman R, Langenbuch M, Lan M, Robinson G, Ruddock L (2001) The
pancreas-specific protein disulphide-isomerase PDIp interacts with a
hydroxyaryl group in ligands. Biochem J 354: 553-559.
Kokame K, Kato H, Miyata T (2001) Identification of ERSE-II, a new cis-acting
element responsible for the ATF6-dependent mammalian unfolded protein
response. J Biol Chem 276: 9199-9205.
Kuchel H, Langridge P, Mosionek L, Williams K, Jefferies SP (2006) The genetic
control of milling yield, dough rheology and baking quality of wheat. Theor
Appl Genet 112: 1487-1495.
Krzywicka A, Beisson J, Keller AM, Cohen J, Jerka-Dziadosz M, Klotz C (2001)
KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia
reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs)
conserved from fission yeast to man. Mol Microbiol 42: 257-267.
Kumari S, Singh P, Singla-Pareek S, Pareek A (2009) Heterologous expression of a
salinity and developmentally regulated rice cyclophilin gene (OsCyp2) in E. coli
and S. cerevisiae confers tolerance towards multiple abiotic stresses. Mol
Biotech 42: 195-204.
La Rota M, Sorrells ME (2004) Comparative DNA sequence analysis of mapped wheat
ESTs reveals the complexity of genome relationships between rice and wheat.
Funct Integr Genomics 4: 34-46.
LaMantia M, Miura T, Tachikawa H, Kaplan H, Lennarz W, Mizunaga T (1991)
Glycosylation site binding protein and protein disulfide isomerase are identical
and essential for cell viability in yeast. Proc Natl Acad Sci USA 88: 4453-4457.
238
Landrieu I, Veylder L, Fruchart J, Odaert B, Casteels P, Portetelle D, Montagu M, Inzé
D, Lippens G (2000) The Arabidopsis thaliana PIN1At gene encodes a single-
domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase. J Biol
Chem 275: 10577-10581.
Langridge P, Lagudah E, Holton T, Appels R, Sharp P, Chalmers K (2001) Trends in
genetic and genome analyses in wheat: A review. Aust J Agric Res 52: 1043-
1077
Larkins B, Hurkman W (1987) Synthesis and deposition of zein in protein bodies of
maize endosperm. Plant Physiol 62: 256-263.
Laudencia C, Stamova B, Lazo G, Cui X, Anderson O (2006) Analysis of the wheat
endosperm transcriptome. J Appl Genet 47: 287-302.
Laxa M, Konig J, Dietz K, Kandlbinder A (2007) Role of the cysteine residues in
Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase
and redox-related functions. Biochem J 401: 287-297.
Leah R, Skriver K, Knudsen S, Ruud-Hansen J, Raikhel NV, Mundy J (1994)
Identification of an enhancer/silencer sequence directing the aleurone-specific
expression of a barley chitinase gene. Plant J 6: 579-89.
Lee J, Hofhaus G, Lisowsky T (2000) Erv1p from Saccharomyces cerevisiae is a FAD-
linked sulfhydryl oxidase. FEBS J 477: 62-66
Levanony H, Rubin R, Altschuler Y, Galili G (1992) Evidence for a novel route of
wheat storage proteins to vacuoles. J Cell Biol 119: 1117-1128.
Leverson J, Ness S (1998) Point mutations in v-Myb disrupt a cyclophilin-catalysed
negative regulatory mechanism. Mol Cell 1: 203-211.
Li QB and Guy CL (2001) Evidence for non-circadian light/dark-regulated expression
of Hsp70s in spinach leaves. Plant Physiol 125: 1633-1642.
Li CP, Larkins BA (1996) Expression of protein disulfide isomerase is elevated in the
endosperm of the maize floury-2 mutant. Plant Mol Biol 30: 873-882.
Li H, He Z, Lu G, Sung C, Alonso J, Ecker J, Luan S (2007) A WD40 domain
cyclophilin interacts with histone H3 and functions in gene repression and
organogenesis in Arabidopsis. Plant cell 19: 2403-2416.
239
Li Y, Song Y, Zhou R, Branlard G, Jia J (2009) Detection of QTLs for bread-making
quality in wheat using a recombinant inbred line population. Plant Breeding 128:
235-243.
Lillemo M, Marco CS, Craig MF (2002) Analysis of puroindoline a and b sequences
from Triticum aestivum cv. 'Penawawa' and related diploid taxa. Euphytica126:
321-331.
Lin D, Lechleiter J (2002) Mitochondrial targeted cyclophilin D protects cells from cell
death by peptidyl prolyl isomerization. J Biol Chem 277: 31134-31141.
Lippuner V, Chou I, Scott S, Ettinger W, Theg S, Gasser C (1994) Cloning and
characterization of chloroplast and cytosolic forms of cyclophilin from
Arabidopsis thaliana. J Biol Chem 269: 7863-7868.
Liu F, Rong Y, Zeng L, Zhang X, Han Z (2003) Isolation and characterization of a
novel human thioredoxin-like gene hTLP19 encoding a secretory protein. Gene
315: 71-78.
Lorkovi Z, Lopato S, Pexa M, Lehner R, Barta A (2004) Interactions of Arabidopsis RS
domain containing cyclophilins with SR proteins and U1 and U11 small nuclear
ribonucleoprotein-specific proteins suggest their involvement in Pre-mRNA
splicing. J Biol Chem 279: 33890-33898.
Loussert C, Popineaua Y, Mangavel C (2008) Protein bodies ontogeny and localization
of prolamin components in the developing endosperm of wheat caryopses. J
Cereal Sci 47: 445-456
Lovat P, Corazzari M, Armstrong J, Martin S, Pagliarini V, Hill D, Brown A, Piacentini
M, Birch-Machin M, Redfern C (2008) Increasing melanoma cell death using
inhibitors of protein disulfide isomerases to abrogate survival responses to
endoplasmic reticulum stress. Cancer Res 68: 5363-5369.
Lu D, Christopher D (2006) Immunolocalization of a protein disulfide isomerase to
Arabidopsis thaliana chloroplasts and its association with starch biogenesis.
Plant Sci 167: 1-9.
Lu D, Christopher D (2008a) Endoplasmic reticulum stress activates the expression of a
sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the
response in Arabidopsis thaliana. Mol Genet Genomics 280: 199-210.
240
Lu D, Christopher D (2008b) The effect of irradiance and redox-modifying reagents on
the 52 kDa protein disulfide isomerase of Arabidopsis chloroplasts Biol Plant
52: 42-48.
Luan S, Albers M, Schreiber S (1994a) Light-regulated, tissue-specific immunophilins
in a higher plant. Proc Natl Acad Sci USA 91: 984–988.
Luan S, Lane W, Schreiber S (1994b) pCyP B: A chloroplast-localized, heat shock-
responsive cyclophilin from fava bean. Plant Cell 6: 885-892.
Luan S, Li W, Rusnak F, Assmann S, Schreiber S (1993) Immunosuppressants
implicate protein phosphatase regulation of K+ channels in guard cells. Proc
Natl Acad Sci USA 90: 2202–2206.
Luu T, Bhattacharya P, Chan W (2008) Cyclophilin-40 has a cellular role in the aryl
hydrocarbon receptor signaling. FEBS Letters 582: 3167-3173
Lyles M, Gilbert H (1991) Catalysis of the oxidative folding of ribonuclease A by
protein disulfide isomerase: dependence of the rate on the composition of the
redox buffer. Biochem 30: 613-619.
Ma W, Appels R, Bekes F, Larroque O, Morell MK, Gale KR (2005) Genetic
characterisation of dough rheological properties in a wheat doubled haploid
population: additive genetic effects and epistatic interactions. Theor Appl Genet
111: 410-422.
Manukyan D, Bruehl M, Massberg S, Engelmann B (2008) Protein disulfide isomerase
as a trigger for tissue factor-dependent fibrin generation. Thromb Res 122: 19-
22.
Markus M, Benezra R (1999) Two isoforms of protein disulfide isomerase alter the
dimerization status of E2A proteins by a redox mechanism. J Biol Chem 274:
1040-1049.
Marraccini P, Deshayes A, Pétiard V, Rogers WJ (1999) Molecular cloning of the
complete 11S seed storage protein gene of Coffea arabica and promoter analysis
in transgenic tobacco plants. Plant Physiol Biochem 37: 273-282
Martin J (1995) Thioredoxin - a fold for all reasons. Structure 3: 245-250
McCartney CA, Somers DJ, Lukow O, Ames N, Noll J, Cloutier S, Humphreys DG,
McCallum BD (2006 ) QTL analysis of quality traits in the spring wheat cross
RL4452×`AC Domain'. Plant Breeding 125: 565-575.
241
Meunier L, Usherwood Y, Chung K, Hendershot LM (2002) A subset of chaperones
and folding enzymes form multiprotein complexes in endoplasmic reticulum to
bind nascent proteins. Mol Biol Cell 13: 4456-4469.
Michelet L, Zaffagnini M, Vanacker H, Marechal P, Marchand C, Schroda M, Lemaire
S, Decottignies P (2008) In vivo targets of S-thiolation in Chlamydomonas
reinhardtii. J Biol Chem 283: 21571-21578.
Mizunaga T, Katakura Y, Miura T, Maruyama Y (1990) Purification and
characterization of yeast protein disulfide isomerase. J Biochem 108: 846-851.
Mogelsvang S, Simpson DJ (1998) Changes in the levels of seven proteins involved in
polypeptide folding and transport during endosperm development of two barley
genotypes differing in storage protein localisation. Plant Mol Biol 36: 541-552.
Mok D, Allan R, Carrello A, Wangoo K, Walkinshaw M, Ratajczak T (2006) The
chaperone function of cyclophilin 40 maps to a cleft between the prolyl
isomerase and tetratricopeptide repeat domains. FEBS Lett 580: 2761-2768.
Morison JIL, Lawlor DW (1999) Interactions between increasing CO2 concentration
and temperature on plant growth. Plant Cell Environ 22: 659–682.
Morris R, Sears ER (1967) The cytogenetics of wheat and its relatives, in Quisenberry
K S, Reitz LP (eds), Wheat and wheat improvement, American Society of
Agriculture, Madison, pp.19-87.
Mortillaro M, Berezney R (1998) Matrin CYP, an SR-rich cyclophilin that associates
with the nuclear matrix and splicing factors J Biol Chem. 273: 8183-8192.
Motohashi K, Kondoh A, Stumpp M, Hisabori T (2001) Comprehensive survey of
proteins targeted by chloroplast thioredoxin Proc Nat Acad Sci USA 98: 11224-
11229
Motohashi K, Koyama F, Nakanishi Y, Ueoka-Nakanishi H, Hisabori T (2003)
Chloroplast cyclophilin is a target protein of thioredoxin. J Biol Chem 278:
31848-31852.
Muccilli V, Cunsolo V, Saletti R, Foti S, Masci S, Lafiandra D (2005) Characterization
of B- and C-type low molecular weight glutenin subunits by electrospray
ionization mass spectrometry and matrix-assisted laser desorption/ionization
mass spectrometry. Proteomics 5: 719 - 728.
Nakamura T, Lipton S (2009) Cell death: Protein misfolding and neurodegenerative
diseases. Apoptosis 14: 455-468.
242
Nguyen V, Wallis K, Howard M, Haapalainen A, Salo K, Saaranen M, Sidhu A,
Wierenga R, Freedman R, Ruddock L, Williamson R (2008) Alternative
conformations of the x region of human protein disulphide-isomerase modulate
exposure of the substrate binding b' domain. J Mol Biol 383: 1144-1155.
Noiva R (1999) Protein disulfide isomerase: the multifunctional redox chaperone of the
endoplasmic reticulum. Semin Cell Dev Biol 1999: 481-493
Norgaard P, Westphal V, Tachibana C, Alsoe L, Holst B, Winther J (2001) Functional
differences in yeast protein disulfide isomerases. J Cell Bio 152: 553-562.
NUE (2003) World wheat, corn, and rice production.
(http://nue.okstate.edu/Crop_Information/World_Wheat_Production.htm;
accessed 01 2009).
Obeid M (2008) ERP57 membrane translocation dictates the immunogenicity of tumor
cell death by controlling the membrane translocation of calreticulin. J Immunol
181: 2533-2543
Oh K, Ivanchenko MG, White TJ, Lomax TL (2006) The diageotropica gene of tomato
encodes a cyclophilin: a novel player in auxin signaling. Planta 224: 133-144.
Ondzighi C, Christopher D, Chob E, Chang S, Staehelin L (2008) Arabidopsis protein
disulfide isomerase-5 inhibits cysteine proteases during trafficking to vacuoles
before programmed cell death of the endothelium in developing seeds. Plant Cell
20: 2205-2220
Parkkonen T, Kivirikko KI, Pihlajaniemi T (1988) Molecular cloning of a
multifunctional chicken protein acting as the prolyl 4-hydroxylase beta-subunit,
protein disulphide-isomerase and a cellular thyroid-hormone-binding protein.
Comparison of cDNA-deduced amino acid sequences with those in other
species. Biochem J 256: 1005–1011.
Payne P (1987) Genetics of wheat storage proteins and the effect of allelic variation on
bread-making quality. Ann Rev Plant Physiol 38: 141-153.
Peleg Z, Cakmak I, Ozturk L, Yazici, A, Yan J, Budak H, Korol AB, Fahima T, Saranga
Y (2009) Quantitative trait loci conferring grain mineral nutrient concentrations
in durum wheat × wild emmer wheat RIL population. Theor Appl Genet 119:
353-369.
Pellé R, McOdimbaa F, Chuma F, Wasawo D, Pearson TW, Murphy NB (2002) The
African trypanosome cyclophilin A homologue contains unusual conserved
243
central and N-terminal domains and is developmentally regulated. Gene 290:
181-191
Perretant M, Cadalen T, Charmet G, Sourdille P, Nicolas P, Boeuf C, Tixier M,
Branlard G, Bernard S (2000) QTL analysis of bread-making quality in wheat
using a doubled haploid population. Theor Appl Genet 100: 1167-1175.
Pflügl G, Kallen J, Schirmer T, Jansonius JN, Zurini MGM, Walkinshaw MD (1993) X-
ray structure of a decameric cyclophilin-cyclosporin crystal complex. Nature
361: 91-94.
Pomeranz Y (1988) Chemical composition of kernel structures, in Pomeranz Y (eds),
Wheat: chemistry and technology. AACC, St Paul, MN, USA, pp. 97-158.
Pozniak C, Knox R, Clarke F, Clarke J (2006) Identification of QTL and association of
a phytoene synthase gene with endosperm colour in durum wheat. Theor Appl
Genet 114: 525-537.
Price ER, Jin M, Lim D, Pati S, Walsh CT, McKeon FD (1994) Cyclophilin B
trafficking through the secretory pathway is altered by binding of cyclosporin A.
Proc Natl Acad Sci USA 91: 3931-3935.
Price ER, Zydowsky LD, Jin MJ, Baker CH, McKeon FD, Walsh CT (1991) Human
cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase
with a signal sequence. Proc Natl Acad Sci USA 88: 1903-1907.
Primm TP, Gilbert HF (2001) Hormone binding by protein disulfide isomerase, a high
capacity hormone reservoir of the endoplasmic reticulum. J Biol Chem 276:
281-286.
Prior A, Uhrig J, Heins L, Wiesmann A, Lillig C, Stoltze C, Soll J, Schwenn J (1999)
Structural and kinetic properties of adenylyl sulfate reductase from
Catharanthus roseus cell cultures. Biochim Biophys Acta 1430: 25-38
Pugh BF, Tjian R (1991) Transcription from a TATA-less promoter requires a
multisubunit TFIID complex. Genes Dev 5: 1935-1945.
Quarrie SA, Steed A, Calestani C, Semikhodskii A, Lebreton C, Chinoy C, Steele N,
Pljevljakusi D, Waterman E, Weyen J, Schondelmaier J, Habash DZ, Farmer P,
Saker L, Clarkson DT, Abugalieva A, Yessimbekova M, Turuspekov Y,
Abugalieva S, Tuberosa R, Sanguineti MC, Hollington PA, Aragués R, Royo A,
Dodig D (2004) A high-density genetic map of hexaploid wheat (Triticum
aestivum L.) from the cross Chinese Spring x SQ1 and its use to compare QTLs
for grain yield across a range of environments. Theor Appl Genet 110: 865-880.
244
Ramos M, Mares R, Magana P, Ortega J, Cornejo-Bravoa J (2008) In silico
identification of the protein disulfide isomerase family from a protozoan
parasite. Comput Biol Chem 32: 67-71.
Rancy P, Thorpe C (2008) Oxidative protein folding in vitro: a study of the cooperation
between quiescin-sulfhydryl oxidase and protein disulfide isomerase. Biochem
47: 12047-12056.
Rascher C, Pahl A, Pecht A, Brune K, Solbach W, Bang H (1998) Leishmania major
parasites express cyclophilin isoforms with an unusual interaction with
calcineurin. Biochem J. 334: 659-667.
Ratajczak T, Carrello A, Mark P, Warner B, Simpson R, Moritz R, House A (1993) The
cyclophilin component of the unactivated estrogen receptor contains a
tetratricopeptide repeat domain and shares identity with p59 (FKBP59). J Biol
Chem 268: 13187-13192.
Ray S, Anderson J, Urmeev F, Goodwin S (2003) Rapid induction of a protein disulfide
isomerase and defense-related genes in wheat in response to the hemibiotrophic
fungal pathogen Mycosphaerella graminicola. Plant Mol Biol 53: 701-714.
Reddy AR, Chaitanya KV, Vivekanandan M (2004) Drought-induced responses of
photosynthesis and antioxidant metabolism in higher plants. J Plant Physiol 161:
1189-1202.
Roden LT, Miflin BJ, Freedman RB (1982) Protein disulphide-isomerase is located in
the endoplasmic reticulum of developing wheat endosperm. FEBS Lett 138:
121-124.
Romano P, Edvardsson A, Ruban A, Andersson B, Vener A, Gray J, Horton P (2004b)
Arabidopsis AtCYP20-2 is a light-regulated cyclophilin-type peptidyl-prolyl cis-
trans isomerase associated with the photosynthetic membranes. Plant Physiol
134: 1244-1247.
Romano PGN, Horton P, Gray JE (2004a) The Arabidopsis cyclophilin gene family.
Plant Physiol 134: 1268-1282.
Rycyzyn M, Clevenger C (2002) The intranuclear prolactin/cyclophilin B complex as a
transcriptional inducer. Proc Natl Acad Sci USA 99: 6790-6795.
Saito T, Niwa Y, Ashida H, Tanaka K, Kawamukai M, Matsuda H, Nakagawa T (1999)
Expression of a gene for cyclophilin which contains an amino-terminal
endoplasmic reticulum-targeting signal. Plant Cell Physiol 40: 77-87
245
Saito Y, Kishida K, Takata K, Takahashi H, Shimada T, Tanaka K, Morita S, Satoh S,
Masumura T (2009) A green fluorescent protein fused to rice prolamin forms
protein body-like structures in transgenic rice. J Exp Biol 60: 615-627.
Salamini F, Ozkan H, Brandolini A, Schafer-Pregl R, Martin W (2002) Genetics and
geography of wild cereal domestication in the near east Genetics 3: 429-441.
Sambrook J, Russell DW (2001) 'Molecular Cloning: A Laboratory Manual.' (Cold
Spring Harbor Laboratory Press, New York)
Sanmartín M, Ordóñez A, Sohn E, Robert S, Sánchez-Serrano J, Surpin M, Raikhel N,
Rojo E (2007) Divergent functions of VTI12 and VTI11 in trafficking to storage
and lytic vacuoles in Arabidopsis. Proc Nat Acad Sci USA 104: 3645-3650
Sargsyana E, Baryshev M, Backlund M, Sharipo A, Mkrtchian S (2002) Genomic
organization and promoter characterization of the gene encoding a putative
endoplasmic reticulum chaperone, ERp29 Gene 285: 127-139.
Schmid F, Grafl R, Wrba A, Beintema J (1986) Role of proline peptide bond
isomerization in unfolding and refolding of ribonuclease. Proc Natl Acad Sci
USA 83: 872-876.
Schonbrunner E, Mayer S, Tropschug M, Fischer G, Takahashi N & Schmid F (1991)
Catalysis of protein folding by cyclophilins from different species. J Biol Chem
266: 3630-3635.
Sears ER (1966) Nullisomic-tetrasomic combination in hexaploid wheat, in Riley R,
Lewis KR (eds), Chromosome manipulations and plant genetics, Oliver and
Body, London, pp. 29-45.
Sears ER (1969) Wheat Cytogenetics. Annu Rev Genet 3: 451-468.
Semenov MA, Halford NG (2009) Identifying target traits and molecular mechanisms
for wheat breeding under a changing climate. J Exp Bot 60: 2791-2804.
Serrato A, Guilleminot J, Meyer Y, Vignols F (2008) AtCXXS: atypical members of the
Arabidopsis thaliana thioredoxin h family with a remarkably high disulfide
isomerase activity. Physiol Plant 133: 611-622.
Shapiguzov A, Edvardsson A, Vener A (2006) Profound redox sensitivity of peptidyl-
prolyl isomerase activity in Arabidopsis thylakoid lumen. FEBS Letters 580:
3671-3676.
246
Sharma A, Singh P (2003) Effect of water stress on expression of a 20 kD cyclophilin-
like protein in drought susceptible and tolerant cultivars of sorghum. J Plant
Biochem Biotechnol 12: 77-80.
Sharma A, Singh P (2006) Peptidyl prolyl cis-trans isomerase activity: A potential
marker for water stress tolerance in sorghum (Sorghum bicolor). Physiol Mol
Biol Plant 12: 91-94.
Sharma A, Wajapeyee N, Yadav V, Singh P (2004) Stress-induced changes in peptidyl-
prolyl cis-trans isomerase activity of Sorghum bicolor. Biol Plantarum 47: 367-
371.
Sheldon PS, Venis MA (1996) Purification and characterization of cytosolic and
microsomal cyclophilins from maize (Zea mays). Biochem J 315: 965-970.
Shewry P (1999) The synthesis, processing, and deposition of gluten proteins in the
developing wheat grain. Cereal Foods World 44: 587-589.
Shewry P (2009) Wheat. J Exp Biol 60: 1537-1553
Shewry P, Napier J, Tatham A (1995) Seed storage proteins: structures and
biosynthesis. Plant Cell 7: 945-956.
Shewry PR, Halford NG (2002) Cereal seed storage proteins: structures, properties and
role in grain utilization. J Exp Bot 53: 947-958.
Shewry PR, Halford NG, Belton PS, Tatham AS (2002) The structure and properties of
gluten: an elastic protein from wheat grain. Phil Trans R Soc Lond B 357: 133-
142.
Shewry PR, Halford NG, Tatham AS, Popineau Y, Lafiandra D, Belton PS (2003) The
high molecular weight subunits of wheat glutenin and their role in determining
wheat processing properties. Adv Food Nutr Res 45: 219-230.
Shimoni Y, Segal C, Zhu XZ, Calili C (1995a) Nucleotide sequence of a wheat cDNA
encoding protein disulfide isomerase. Plant Physiol. 107: 281.
Shimoni Y, Zhu X, Levanony H, Segal G, Galili G (1995b) Purification,
characterization, and intracellular localization of glycosylated protein disulfide
isomerase from wheat grains. Plant Physiol 108: 327-335.
Shorrosh B, Subramaniam J, Schubert K, Dixon R (1993) Expression and localization
of plant protein disulfide isomerase. Plant Physiol 103: 719-726.
247
Short D, Heron I, Birse-Archbold J, Kerr L, Sharkey J, McCulloch J (2007) Apoptosis
induced by staurosporine alters chaperone and endoplasmic reticulum proteins:
Identification by quantitative proteomics. Proteomics 7: 3085-3096
Simeone MC, Gedye KR, Mason-Gamer R, Gill BS, Morris, CF (2006) Conserved
regulatory elements identified from a comparative puroindoline gene sequence
survey of Triticum and Aegilops diploid taxa. J Cereal Sci 44: 21-33.
Simpson CG, Clark G, Davidson D, Smith P, Brown JWS (1996) Mutation of putative
branchpoint consensus sequences in plant introns reduces splicing efficiency
Plant J 9: 369-380.
Sirpio S, Khrouchtchov A, Allahverdiyeva Y, Hansson M, Fristedt R, Vener A, Scheller
H, Jensen P, Haldrup A, Aro EM (2008) AtCYP38 ensures early biogenesis,
correct assembly and sustenance of photosystem II. Plant J 55: 639-651
Sliskovic I, Raturi A, Mutus B (2005) Characterization of the S-denitrosation activity of
protein disulfide isomerase. J Biol Chem 280: 8733-8741.
Smith M, Willmann M, Wu G, Berardini T, Moller B, Weijers D, Poethig R (2009)
Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc Nat Acad
Sci USA 106: 5424-5429.
Smith T, Ferreira LR, Hebert C, Norris , KJ, Sauk JJ, (1995) Hsp47 and cyclophilin B
traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate
vesicles. J Biol Chem 270: 18323-18328.
Song J, Wang C (1995) Chaperone-like activity of protein disulfide-isomerase in the
refolding of rhodanese. Eur J Biochem 231: 312-316.
Stålberg K, Ellerstöm M, Ezcurra I, Ablov S, Rask L (1996) Disruption of an
overlapping E-box/ABRE motif abolished high transcription of the napA
storage-protein promoter in transgenic Brassica napus seeds. Planta 199: 515-
519.
Sykes K, Gething M, Sambrook J (1993) Proline isomerases function during heat shock.
Proc Natl Acad Sci USA 90: 5853–5857.
Takaki Y, Muta T, Iwanaga S (1997) A peptidyl-prolyl cis/trans-isomerase (Cyclophilin
G) in Regulated Secretory Granules. J Biol Chem 272: 28615-28621.
Takemoto Y, Coughlan SJ, Okita TW, Satoh H, Ogawa M, Kumamaru T (2002) The
rice mutant esp2 greatly accumulates the glutelin precursor and deletes the
protein disulfide isomerase. Plant Physiol. 128: 1212-1222.
248
Theriault Y, Logan T, Meadows R, Yu L, Olejniczak E, Holzman T, Simmer R, Fesik S
(1993) Solution structure of the cyclosporin A/cyclophilin complex by NMR.
Nature 361: 88-91.
Thorpe C, Hoober K, Raje S, Glynn N, Burnside J, Turi G, Coppock D (2002)
Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in
eukaryotes. Arch Biochem Biophys 405: 1-12.
Tosi P, Parker M, Gritsch CS, Carzaniga R, Martin B, Shewry PR (2009) Trafficking of
storage proteins in developing grain of wheat. J Exp Biol 60: 979-991.
Trebitsh T, Danon A (2001) Translation of chloroplast psbA mRNA is regulated by
signals initiated by both photosystems II and I. Proc Nat Acad Sci USA 98:
12289-12294.
Tremmel D, Duarte M, Videira A, Tropschug M (2007) FKBP22 is part of
chaperone/folding catalyst complexes in the endoplasmic reticulum of
Neurospora crassa. FEBS Lett 581: 2036-2040
Triboi E, Martre P, Triboi-Blondel A (2003) Environmentally-induced changes in
protein composition in developing grains of wheat are related to changes in total
protein content. J Exp Bot 54: 1731-1742.
Ushioda R, Hoseki J, Araki K, Jansen G, Thomas D, Nagata K (2008) ERdj5 is required
as a disulfide reductase for degradation of misfolded proteins in the ER. Science
321: 569-572.
van Lith M, Hartigan N, Hatch J, Benham A (2005) PDILT: A divergent testis-specific
PDI with a non-classical SXXC motif that engages in disulfide dependent
interactions in the endoplasmic reticulum. J Biol Chem 280: 1376-1383.
Vicente-Carbajosa J, Moose SP, Parsons RL, Schmidt RJ (1997) A maize zinc-finger
protein binds the prolamin box in zein gene promoters and interacts with the
basic leucine zipper transcriptional activator Opaque2. Proc Natl Acad Sci USA
94: 7685-90.
Vitale A, Ceriotti A (2004) Protein quality control mechanisms and protein storage in
the endoplasmic reticulum. A conflict of interests? Plant Physiol 136: 3420-
3426,.
Vitale A, Galili G (2001) The endomembrane system and the problem of protein sorting
Plant Physiol 125: 115-118
249
Vitu E, Gross E, Greenblatt H, Sevier C, Kaiser C, Fass D (2008) Yeast Mpd1p reveals
the structural diversity of the protein disulfide isomerase family. J Mol Biol 384:
631-640
Vuori K, Pihlajaniemi R, Myllyla T, Kivirikko KI (1992) Site-directed mutagenesis of
human protein disulphide isomerase: effect on the assembly, activity and
endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera
frugiperda insect cells. EMBO J. 11: 4213-4217.
Wadahama H, Kamauchi S, Nakamoto Y, Nishizawa K, Ishimoto M, Kawada T, Urade
R (2008) A novel plant protein disulfide isomerase family homologous to animal
P5 – molecular cloning and characterization as a functional protein for folding of
soybean seed-storage proteins. FEBS J 275: 399 - 410.
Walsh C, Zydowsky L, McKeon F (1992) Cyclosporin A, the cyclophilin class of
peptidyl prolyl isomerases, and blockade of T cell signal transduction. J Biol
Chem 267: 13115-13118.
Wang H, Boavida L, Ron M, McCormick S (2008) Truncation of a protein disulfide
isomerase, PDIL2-1, delays embryo sac maturation and disrupts pollen tube
guidance in Arabidopsis thaliana. Plant Cell 20: 3300-3311
Wang L, Li S, Sidhu A, Zhu L, Liang Y, Freedman RB, Wang C (2009) Reconstitution
of human Ero1-Lα/protein-disulfide isomerase oxidative folding pathway in
vitro position-dependent differences in role between the a and a′ domains of
protein-disulfide isomerase. J Biol Chem 284: 199-206.
Wang P, Heitman J (2005) The cyclophilins. Genome Biol 6: 226-232.
Wang Y, Han R, Zhang W, Yuan Y, Zhang X, Long Y, Mi H (2008) Human CyP33
binds specifically to mRNA and binding stimulates PPIase activity of hCyP33.
FEBS Lett 582: 835-839
Wells W, Xu D, Yang Y, Rocque P (1990) Mammalian thioltransferase (glutaredoxin)
and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol
Chem 265: 15361-15364.
Wetterau JR, Combs KA, McLean LR, Spinner SN, Aggerbeck LP (1991) Protein
disulfide isomerase appears necessary to maintain the catalytically active
structure of the microsomal triglyceride transfer protein. Biochem 30: 9728-
9735.
250
Wheeler TR, Craufurd PQ, Ellis RH, Porter JR, Prasad PVV (2000) Temperature
variability and the yield of annual crops. Agric Ecosys Environ 82: 159-167.
Wiborg J, O'Shea C, Skriver K (2008) Biochemical function of typical and variant
Arabidopsis thaliana U-box E3 ubiquitin-protein ligases. Biochem J 413: 447-
457
Wilkinson B, Gilbert H (2004) Protein disulfide isomerase. Biochim Biophys Acta
1699: 35-44.
Wilson R, Lees J, Bulleid N (1998) Protein disulfide Isomerase acts as a molecular
chaperone during the assembly of procollagen. J Biol Chem 273: 9637-9643.
Winter D, Vinegar B, Nahal H, Ammar R, Wilson G, Provart N (2007) An “electronic
fluorescent pictograph” browser for exploring and analyzing large-scale
biological data sets. PLoS ONE 2: e718.
Woycechowsky K, Raines R (2000) Native disulfide bond formation in proteins. Curr
Opin Chem Biol 4: 533-539.
Wrobel RL, OBrian GR, Boston RS (1997) Comparative analysis of BiP gene
expression in maize endosperm. Gene 204: 105-113.
Wu H, Wensley E, Bhave M (2009) Identification and analysis of genes encoding a
novel ER-localised Cyclophilin B in wheat potentially involved in storage
protein folding. Plant Sci 176: 420-432.
Yao Y, Zhou Y, Wang C (1997) Both the isomerase and chaperone activities of protein
disulfide isomerase are required for the reactivation of reduced and denatured
acidic phospholipase A2. EMBO J 16: 651-658.
Yoshida H, Haze K, Yanagi H, Yura T, Mori K (1998) Identification of the cis-acting
endoplasmic reticulum stress response element responsible for transcriptional
induction of mammalian glucose-regulated proteins. Involvement of basic
leucine zipper transcription factors. J Biol Chem. 273: 33741-33749.
Zwart RS, Thompson JP, Milgate AW, Bansal UK, Williamson PM, Raman H, Bariana
HS (2010) QTL mapping of multiple foliar disease and root-lesion nematode
resistances in wheat. Mol Breeding. 10.1007/s11032-009-9381-9.
Zydowsky LD, Etzkorn FA, Chang HY, Ferguson SB, Stolz L, Ho SI, Walsh CT (1992)
Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase
activity from cyclosporin A binding and calcineurin inhibition. Protein Sci. 1:
1092-109.
251
Appendices Appendix I Tentative Consensus (TC) sequence (TC264488) for primers design
Primer sequences designed for isolation of Cyclophilin B in wheat are shaded. Forward arrows indicate forward primers; and reverse arrows indicate reverse primers. Start codon (S.C.) and stop codon (S.T.) are bolded and underlined.
252
Appendix II Sequences data from individual clones and /or direct PCR products in wheat
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ATACGATCCGAGATGGCGATGAGGGCGTGGAGGAGGAGCGCGGCGGCGAGGGCCCCGGCGCACCTGTGCCTGTGGCTGGCGCTCGTCGCCGCCACCCTGGTGCTCGCCCAGGTGCTCATCCACCTCGTCCGTCTTCCGCGAACCCATCCC
TaA1 .........A............................................................................................................................................
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ......................................................................................................................................C..........C...T
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 .................................C....................................................................................................A...............
TdA2 ......................................................................................................................................................
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ......................................................................................................................................C..........C...T
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 .................................C....................................................................................................A...............
TuA ......................................................................................................................................C..........C...T
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ...............................................................................................................---------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 CTTACTTTTCCGCTTGCCGATTTGTTCGCCGATCTGATCGTTTCTCTGATACGGTTAATCGTTGCCCTCGCGCTGATGTCTGAACCTGTCTGCCGCGGATCCGGATGGCTTATATGTCTTCGGCGCTGCTGATTTGTTAGGGTTTGGTGT
TaA1 ......................................................................................................................................................
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ...TAC.......CGCT.CCCCCTC....TCC..C.TG..CCGA......-..T.CCTCT.C.A.GA.T..T.----.CTGAT.T....T...........G..GC..A...G....G..T..G.T.....G.C............A.--
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 .........................................................................................................C...........------------------...........A...
TdA2 ......................................................................................................................................................
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ...TAC.......CGCT.CCCCCTC....TCC..C.TG..CCGA......-..T.CCTCT.C.A.GA.T..T.----.CTGAT.T....T...........G..GC..A...G....G..T..G.T.....G.C............A.--
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 .........................................................................................................C...........------------------...........A...
TuA ...TAC.......CGCT.CCCCCTC....TCC..C.TG..CCGA......-..T.CCTCT.C.A.GA.T..T.----.CTGAT.T....T...........G..GC..A...G....G..T..G.T.....G.C............A.--
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GGTTGGTGTCTCTGTATGGGATGCGTACTTACGATTTTCTTCCTTGTTTTCAGGGCAAGAAATCCAACCTGTCTGAGGTGACGCACAAGGTCTACTTCGACATCGAGATCGACGGCAAGCCCGCAGGTCTGTGCTGAACCCCTGGCCACA
TaA1 ......................................................................................................................................................
TaB1 ---------------------------------------------------------------------------------.....................................................................
TaB6 ---------------------------------------------------------------------------------.....................................................................
TaA4 --C........G.......-......G.....A........TG.G....C.....T.....G..G........G.............................T.....................................G....A...
TaB2 ---------------------------------------------------------------------------------......................T.....................................G....A...
TaA5 ......................................................................T............................................................................G..
TdA2 ......................................................................................................................................................
TdB1 ---------------------------------------------------------------------------------.....................................................................
TdA1 --C........G.......-......G.....A.........G.G....C.....T.....G..G........G.............................T.G...................................G....A...
TdB3 ---------------------------------------------------------------------------------......................T.G...................................G....A...
Aet1 ......................................................................T............................................................................G..
TuA --C........G.......-......G.....A........TG.G....C.....T.....G..G........G.............................T.....................................G....A...
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 -----------------------------------------------------.........................................................................------------------------
Exon I
S.T.
Intron I
HaeIII
Exon II Intron II
A
HaeIII
253
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 AATTTGTTTTCATATCAGCGGGATGTATCATCCCAGTATGGGGCAGGCAGTAGACTGCTACTAATACAACCGATATTGCATGCCTGATTCCTAGTCCTAGCCTAGAAAGCACGGTTTGTAGTTACACTTCGAGTGATTCCTTTTGATTAG
TaA1 ......................................................................................................................................................
TaB1 ......................................................................................................................................................
TaB6 ......................................................................................................................................................
TaA4 G....A......G.......C...........T...........G.A...............G.............C...............G.C...............C..............G.G..........A......C....
TaB2 G....A......G.......C...........T...........G.A...............G.............C...............G.C...............C..............G.G..........A......C....
TaA5 G....A......G.......C...............C.........A...............G........A....C.........T....------.G...........C.............G..G.T...............C..T.
TdA2 ......................................................................................................................................................
TdB1 ......................................................................................................................................................
TdA1 G....A......G..T....C...........T...........G.A...............G.............C...............G.C............G..C..............G.G..........A......C....
TdB3 G....A......G..T....C...........T...........G.A...............G.............C...............G.C............G..C..............G.G..........A......C....
Aet1 G....A......G.......C...............C.........A...............G........A....C.........T....------.G...........C.............G..G.T...............C..T.
TuA GG...A......G.......C...........T...........G.A...............G.............C...............GTC...............C..............G.G..........A......C....
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GATCGTTGAAAAA--TGTGAGATTGACATTGAAATTGAATTTCCATGTTTAGATGGCTGCACTGCACCTAATTTTGTTTTATGTTTA---GTGAGAAAAATATTCACTGGTGTGCTATGCATGTACGTTCTTAATGGTTTAACTCCTTTT
TaA1 .............--........................................................................---............................................................
TaB1 .............--........................................................................---............................................................
TaB6 .............--........................................................................---............................................................
TaA4 .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
TaB2 .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
TaA5 .............AT........C..................T...................C..G.....................TAA............................T......T.......T..--.GC.TCT....C
TdA2 ......C.....G--........................................................................---............................................................
TdB1 ......C.....G--........................................................................---............................................................
TdA1 .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
TdB3 .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
Aet1 .............AT........C..................T...................CA.G.....................TAA............................T......T.......T..--.GC.TCT....C
TuA .......T.---------------------..GGC.......T............C................CA.............---..........................------------.......A--------------
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 CATTGTTTGATTGATCCGCTGGCATTTAAATTGATTCTGAATTGTCATAGGTCGGGTTGTCATGGGACTTTTCGGCAAGGCTGTTCCTAAAACCGCAGGTATCATCTCCTTACTGTTTGAGTTAAAAACCATGTACTCATCTATTGGATG
TaA1 ......................................................................................................................................................
TaB1 ......................................................................................................................................................
TaB6 ......................................................................................................................................................
TaA4 ----..............T..........................................................................................C.G.....C................................
TaB2 ----..............T..........................................................................................C.G.....C................................
TaA5 ..................G..................................................................................................C......C...............T..C......
TdA2 ...................................................................................................................................C..................
TdB1 ...................................................................................................................................C..................
TdA1 ----..............T..........................................................................................C.G.....C................................
TdB3 ----..............T..........................................................................................C.G.....C................................
Aet1 ..................G..................................................................................................C......C...............T..C......
TuA ----..............T..........................................................................................C.G.....C................................
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 --------------------------------------------------................................................----------------------------------------------------
Exon III Intron III
HaeIII
254
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ACAGAAATGCTAATGGCTTGATATCTTTTGTTCTACAGAGAACTTCAGAGCACTCTGCACAGGTATGCTTCCCTACCACTTGCGCTGTTTTCTATATGTTATAAGCGTTATGCCGTCTGCTTGTTGAAGTTTCTAGATGATTAGTCTGAT
TaA1 ......................................................................................................................................................
TaB1 ......................................................................................................................................................
TaB6 ......................................................................................................................................................
TaA4 ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
TaB2 ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
TaA5 ..................C.....................................................G...T............-----------.C.TT.T...C...............G......C................
TdA2 ......................................................................................................................................................
TdB1 ......................................................................................................................................................
TdA1 ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
TdB3 ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
Aet1 ........................................................................G...T............-----------.C.TT.T...C...............G......C................
TuA ...............................C.................................................AT........T.G.......C...........T....A......T......T.G...............
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 --------------------------------------.............G..........----------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GGCAAGGCTGGCTATTGGAATAAAAT-TTGCTCTATACAATCACAGTTGCTTTGTTATTTTGGCTCACATCTTCAAGTAATTTAGCCAATCAAATGTAATTGGCTTCCTCAGAAAGTCAAAACATAATTGTACAGTGGAAATTATATACT
TaA1 ..........................-...........................................................................................................................
TaB1 ..........................-...........................................................................................................................
TaB6 ..........................-...........................................................................................................................
TaA4 ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
TaB2 ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
TaA5 ............GT............-C........C....T..T...A...-............A--..........T................A......C...........................C...A...........----
TdA2 ..........................-..............................................A............................................................................
TdB1 ..........................-..............................................A............................................................................
TdA1 ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
TdB3 ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
Aet1 ............GT............-C........C....T..T...A...-............A--..........T................A......C...........................C...A...........----
TuA ............C............AA.........TT...................C.............................T.......A.G.............................C..C......-........----
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GTAACTAATTTGATGCATGCTATGCTTGTAACATGGAAACGTTG--CTTGCCTTTTTCTTATAAAAGAAGGAAGCTATCTGTATTGTTCCCTTGTTATTTCAAATCCAAGTATACCTTTCTAGACACGACA---TAGAGAGAATGGTGGC
TaA1 ............................................--.....................................................................................---................
TaB1 ............................................--...................................................C.................................---................
TaB6 ............................................--.....................................................................................---................
TaA4 -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
TaB2 -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
TaA5 -.....C.............................C..A.C..TG...A........C.TG..G........T...C..........T...CA.................T....A..........A.G.ATG................
TdA2 ............................................--.....................................................................................ACG................
TdB1 ............................................--.....................................................................................ACG................
TdA1 -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
TdB3 -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
Aet1 -.....C.............................C..A.C..TG...A........C.CG..G........T...C..........T...CA.................T....A..........A.G.ATG................
TuA -...................................C..A.C..TG...A........C.TG..G............C..........T...CA.................T....A..........A.G.ACG..............A.
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon IV Intron IV
HaeIII
255
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 AGCCTGTTTGCCTGCAAATTTTTGGGCATGATCTGTTTAAATCCCAAGCTCCCTAGAGCATCATTGAAGGGTTCTAAGTTGTATGTTCTACACAGTCTCTAGTAGTAGCCCTGTGTGTGGA-TTTTGATTGTTCATCCCTCCTCTACCAT
TaA1 .........................................................................................................................-............................
TaB1 .........................................................................................................................-............................
TaB6 .........................................................................................................................-............................
TaA4 ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
TaB2 ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
TaA5 .........T........G.......G..C............T....A.C....C......A........-.....T......A.....G...G.....A..G..............CA..A..................TG--------
TdA2 .........................................................................................................................-............................
TdB1 .........................................................................................................................-............................
TdA1 ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
TdB3 ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
Aet1 .........T........G.......G..C............T....A.C....C......A........-.....T......A.....G...G.....A..G..............CA..A..................TG--------
TuA ..................G.......G...............T...........C......A.......T......T......A.-----------...A..G..............CAT.A..................TG--------
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GGTTCTAAAAGAAATCCCTCTACCATACAAGTGCCCAAATATGAATGTCAATAAGAAAAAA-GAGAAGAAAT-AGATAACTGGTTGAATTTGTAGTTGGCTGGTGCCTAGCTGAATATATC--AGAGTATTTTACTTCATGGAAA--AAG
TaA1 .............................................................-..........-................................................A--.....................--...
TaB1 .............................................................-..........-................................................A--.....................--...
TaB6 .............................................................-..........-................................................A--.....................--...
TaA4 -----------------.................-T.............T...T.......A..........G.AG...........G..C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
TaB2 -----------------.................-T.............T...T.......A..........G.AG...........G..C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
TaA5 -----------------..................T.................T.......-..........T.AG.......C...G..C.......T..A........-------.C.AA--.TA..............A...GA...
TdA2 ..........A........G.........................................-..........-................................................A--.....................--...
TdB1 ..........A........G.........................................-..........-................................................A--.....................--...
TdA1 -----------------.................-T.............T...T.......A..........G.AG...........G..C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
TdB3 -----------------.................-T.............T...T.......A..........G.AG...........G..C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
Aet1 -----------------..................T.................T.......-..........T.AG.......C...G..C.......T..A........-------.C.AA--.TA..............A...GA...
TuA -----------------.................-T.............T...T.......A..........G.AG..............C.......T..A........TCT.....G..AA..TA......G.......A.C.AA...
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GTGGATAAATGTTGTCATACTCTTCATA--------TTCCACCATAGAGTATTACGAAAGCCTATCATTATTTACTGCATATATCATTATATTCAGAATATCTGCTTTATTAGTACTACAGTCTGTATGGTAGCTT--------GATATC
TaA1 ............................--------....................................................................................................--------......
TaB1 ............................--------......................G.............................................................................--------......
TaB6 ............................--------....................................................................................................--------......
TaA4 ........C...C..T.C...--.....--------......................G.............................G....................C..................T.......CTCTCTAT......
TaB2 ........C...C..T.C...--.....--------......................G.............................G....................C..................T.......CTCTCTAT......
TaA5 ........C...C..T.C..........AGTTCATA.....A.........................C..............-................................A....A.......A.......CTCTCTAT...G..
TdA2 ............................--------....................................................................................................--------......
TdB1 ............................--------....................................................................................................--------......
TdA1 ........C...C..T.C...--.....--------......................G.............................G....................C..................T.......CTCTCTAT......
TdB3 ........C...C..T.C...--.....--------......................G.............................G....................C..................T.......CTCTCTAT......
Aet1 ........C...C..T.C..........AGTTCATA.....A.........................C..............-................................A....A.......A.......CTCTCTAT...G..
TuA ........C...C..T.C...--.....--------......................G.............................G....................C.............C....T.......CTCTCTAT......
TuD2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
HaeIII
256
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 TGCCACTGGTTGCTGACCATGCCCCACTTAATTGTCATACAGGTGAGAAAGGCATGGGCAACAGCGGCAAACCTCTCCACTACAAGGGGAGTTCGTTCCACAGAATTATCCCCAGCTTCATGATCCAAGGAGGGGACTTCACTCTTGGCG
TaA1 .......................................................................................................-----------------------------------------------
TaB1 ......................................................................................................................................................
TaB6 ......................................................................................................................................................
TaA4 ...T.G.....................................A..........................G................................-----------------------------------------------
TaB2 ...T.G.....................................A..........................G...............................................................................
TaA5 ...T.G..........................C..........A........T..................................................-----------------------------------------------
TdA2 .....G.................................................................................................-----------------------------------------------
TdB1 .....G................................................................................................................................................
TdA1 ...T.G.....................................A.G........................G................................-----------------------------------------------
TdB3 ...T.G.....................................A.G........................G..............................................................T................
Aet1 ...T.G..........................C..........A........T................................................................................C..........C...T.
TuA ...T.G.....................................A..........................G................................-----------------------------------------------
TuD2 --------------------------------------.....A........T.................G..............................................................T................
TaCD4 ------------------------------------------............................................................................................................
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ATGGAAGGGGCGGTGAATCAATCTATGGCACAAAGTTCGCCGATGAGAACTTCAAGCTCAAGCACACTGGACCAGGTAATGATCCGTGTTTCTTGAAATCCAGTTC-CAACTGCAAGTGA-AC-GATAGAATGATAGCCGGGGTTAAGAT
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ..........................................................................................................-.............-..-..........................
TaB6 ..........................................................................................................-.............-..-..........................
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 .........................C.................................................................T..............-............CT..-.G.....A.....G.C..........
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ..........................................................................................................-.............-..-..........................
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 .........................C................................................................................-............CT..-.G.....A.....G.C..........
Aet1 ..........T..............C...........................................................T................T..TG......G...A.CTC.-.T.............CT.......CC
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 .........................C................................................................................-............CT..-.G.....G.....G.C..........
TaCD4 ...........................................................................---------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GTCCTCCTTTTTCATTGACTGGTTGTTGGTGCAGGCTACCTTTCCATGGCCAATGCTGGGAGAGATACCAATGGATCCCAGTTCTTCATCACCACTGTAACCACGAGCTGGTAATT-TTCATGA--CTCTGTGATTGCAGCAGTTTGTAC
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ....................................................................................................................-.......--........................
TaB6 ....................................................................................................................-.......--........................
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ..........................C.........................................................................................-.......TT..T.............T....CG.
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ....................................................................................................................-.......CT..--....................
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ..........................C.........................................................................................-.......TT..T.............T....CG.
Aet1 A.........................C............................................C.............................T..............C....C..TG..T...A..C....T.A.....G.
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 ..........................C.........................................................................................-.......TT..T.......A.....T....CG.
TaCD4 ----------------------------------............................................................................----------------------------------------
Exon V
Intron V
Exon VI Intron VI
257
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 TAAGCCTTT-TATTCTGCTTGAA-------CTTTGTTGTTGATGATCTACATTTTGTATGTTAGGTTGGACGGCAAGCACGTCGTGTTCGGCAAGGTGTTGTCTGGAATGGACGTGGTCTACAAGGTTGAGGCCGAGGGCAAGCAGAACG
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 .........-.............-------........................................................................................................................
TaB6 .........-.............-------........................................................................................................................
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 .G.AT....C.G.......G...-------..C.A...........A....C.C.....TC.........T...............................................................................
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 .........-.............-------........................................................................................................................
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 .G.AT....C.G.......G...-------..C.A...........A....C.C.....TC.........T...............................................................................
Aet1 ...A.....-.G.....T.G.G.GTTGGGA................A....C.C.....TC.........T........................................................C......................
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 .G.AT....C.G.......G-------GAA..CCA...........A....C.C.....TC.........T...............................................................................
TaCD4 ----------------------------------------------------------------......................................................................................
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 GGACGCCAAAGAGCAAGGTTGTCATTGCCGACAGCGGTGAAGTGCCGCTGTGATGAGACCTGAAATGGGCTCTGGTGGAA-GCTACGGTGACTAGTTTTCGTTGTAGAACCGTTACTAGCCGGTCTGTTTTTTGTTTTCGTGTACTTTTG
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ..................................--------------------------------------------------------------------------------------------------------------------
TaB6 ..................................--------------------------------------------------------------------------------------------------------------------
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ..................................--------------------------------------------------------------------------------------------------------------------
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ..................................--------------------------------------------------------------------------------------------------------------------
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ..................................--------------------------------------------------------------------------------------------------------------------
Aet1 ....A...........A...........T........G..................--.......C..T...........A.................C..........G.....T..T....A.-------.A.............CC.
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 .........................C...........A..............................T...........A...................A..............T.GT.G..A.-------...............CC.
TaCD4 ................................................................................-...................................................................C.
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 TGTGAAATTCAGACGACAAGGATTCTC----TTGCTATAAGAAAGTATAATGACTTGCGGATTTACCCGTGATCTGGCTGGCAACTTTGAAGGCATCACACACCACCGT-----------------------------------------
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 ......-..T.....G....TT..T..CTCT..A......A......C..........----...............................T...............-----------------------------------------
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 ......-..T.....G...A.C..T.TCTCT.-A......A..........A......----....T.A....A...A..............TGC..TT.TTATTTTT.CTTGGATGTTTTGCTGGGTTATTTGCCTTGGAGAAAGTGCT
TaCD4 ...........................----...........................----C..............................................-----------------------------------------
Exon VII
HaeIII
S. P.
258
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 GATATGCGCCTTTTGATTTGAACGATTATCGATCGTCAGCAAACAAATATTCTCGCTTAGTACTGCAAGTGCAGCGGCCGATACCTCCCGCCTGGTGATTGTTTTTTTATGAGGACCAATCTCACAGTAGTTTTATACTTGAAAATCTTT
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 AAAGAAACATTTCTGGCTTTACGCTTTAAAAGGTCTGAAAACTGTTGGGAGCACTACCCTATGACCTTTGTGTCGATGAAGCTCCACCACTGCCCGGTAGAGGAGAATCTCCAACAAACCTCCACCACCATAGACCACAGTAGTATTCTT
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 TTTCCCTGGCAGAAAAAGAAATATTCTTTGTTAAAAAAAGAGAAAGAAATATTCTCTTACAATCCTATTTGTTTTTCTTATACGATAATAATCTACGGTCCGCATCTTCTTCTCATACGATTGGTACTATGAATATACGTAAAAAAAGTA
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
259
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 AACAGAGTCCGCGGTTTTAAGAAATAGGAAAAGAAAAGGAGTTATGCAAAGACACGGCGAGCAACCCTAGAAGCCGCATCTTCCACTCAACCAATATATTTTTCTTTCATCTAGTATATCATATTAAAAAAAAGGAGGAATCCTACAATC
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB6 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA4 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaB2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TaA5 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA2 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdA1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TdB3 ------------------------------------------------------------------------------------------------------------------------------------------------------
Aet1 ------------------------------------------------------------------------------------------------------------------------------------------------------
TuA ------------------------------------------------------------------------------------------------------------------------------------------------------
TuD2 AGCATCAGGGCCGCTGGCTCCAGATCGGACGGTGGAGCGGGCAGAGCCGCGCGCCCAGGGGGCCTAATAATTGGCGTCCCCCCATCCGCCCCCGAGCCGCCCCCAAACCCCGTCCCGTCAAACGCAAAGAAGACAAGCGCAGCAAGTAGG
TaCD4 ------------------------------------------------------------------------------------------------------------------------------------------------------
3460 3470 3480 3490
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
TaC1 ----------------------------------------
TaA1 ----------------------------------------
TaB1 ----------------------------------------
TaB6 ----------------------------------------
TaA4 ----------------------------------------
TaB2 ----------------------------------------
TaA5 ----------------------------------------
TdA2 ----------------------------------------
TdB1 ----------------------------------------
TdA1 ----------------------------------------
TdB3 ----------------------------------------
Aet1 ----------------------------------------
TuA ----------------------------------------
TuD2 GTTAGGAGAGCCGACGGCGGCGGCGGCGGCGGAAAGCGAG
TaCD4 ----------------------------------------
260
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) AGAGAATCTTTGTATGTGGTTTGGGGCAGTGAAGCGACCGCGTTGCCCCAGAGTAGGAATGATATTCTCTATGCGGGTGGAGGGCATGTTTTTTCGCTGGTGGAGGGCCCTATTCCCCGTTTCATTTGTGTGTGTTTTCGTTGGTGGAGC
TuCypB-A(P) ------------------------------------------------------------------------------------------------------------------------------------------------------
Rice (P) ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) ACGAATGGAGCTATATATTTGACGAGTCGTTTGGATTCCAGACGGTTTAGGTATCCGGTGGATCTTATCTAGATTTGGTCGGCTTTCGTGGTACTCGGAGATTGTACAACCCC-TTATCGACATCCTTTTCTCCGGGTTGTCAGCATTTC
TuCypB-A(P) ------------------------------------------------------------------------------------------------------------------------------------------------------
Rice (P) -----------------------------------------------------------------------AGAAGCT.AATAACGA.CT.....TACTT.CC..GA.TT..TA.C..T.GAG.ATG.G.GCA..TCA.TAGT..CA.G.
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) CTAGTCTGCATCAACGACTTCCCGACCGTTGCTTCTACAAACTCATGGATTTCAAAAAGTTAGGATCGTCGATGCGAAGGCCTAGAAGCGGCATCAAGCTATGCCCACGACACGCCGCCGATGGATGCGGGACGAAGAAGACTTCGGCTC
TuCypB-A(P) ------------------------------------------------------------------------------------------------------------------------------------------------------
Rice (P) A.T..T..G.G.CTGTTTC.G.T..TTA.AATG.GAC.TGCT.TG..T.A..GTTGT.AAAT.AGAGTGTTC.CTC..TATT.TCCCAT....ATTCAGAT..GAT.T.CA.T..AAG.T-.AT..A.AT.CGA.CAC.ATG.CATAT..
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) CCCTAAGATTTTGAATGTATTTCTATTTTTTCTGCAAGGGTGTCTTTTTATAAGGACCGATGATACTTAATAACGAAGGGAGTAGTAATCACAACAAACGCGTGATACACCTACTTACTAGACCCAACATGATAGAGCTGTAACAAACCC
TuCypB-A(P) ------------------------------------------------------------------------------------------------------------------------------------------------------
Rice (P) .T..TT.....A..T.A.TGAG.GGA..A...CATG.ATTATCAA...G..T.T...T-..A.ATTAA.TA.TTT.TAAA.A..AACT.A.T..AT.GTTTAAA.C..G.A.TT...AGCCTTGT.CA.GA.A.TTCT.T.T.T...AAA
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) AAGTTCTTAATTAACCGCAAGATCTAAATGTATTATTTTCATGAACATAATTCTTGTTTACCCCCATCCGTTTCAAAATAAGTGTTATGGTTTTAGTTCAATACCACGACACTTAGTTTTGAACGGTGGATCAACTCTCTAAAAAGAGTC
TuCypB-A(P) ---------------------------------------------------------------------------------------------------------GA.CTC.AA.T.....C..T....T.TCGT.TGGCTTCC...AGT
Rice (P) ..T..AAAGCG.TGAGCA..T.ATCCGT.TCTA..A..AG.AT..GG.TTAGT...A..GTGAAGT.TG.A..TTT...GGCACG..AAA.AAG.C.AG.C..ATTAAGT.T.A.T.A..T..AAA.TAT.TGTT.T.T-------.AAT
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) AAAATACTTGTGTGCTTCTTTGTGAAGGGTGAAATAGTGTTAGAAGAGAGAGAAAG-AGTTTTTTTGCATAACAGG-GAAAAAGAG-TTAGCAGTAGGGTTTATATTTTGGTACCCACTGAAATTACCGAAATTTCAGAAGTTTGGTGAT
TuCypB-A(P) T.GC.T...A-...TAAAAGC..TCG.CTCA...ACT.T.CT....TTT.T...GTT..AA.C.GGAGGAG.TCA.-A.C.CGCCC-...AT..........G................A......G...........---.........
Rice (P) ...C.T..AAACAAAAAG.A.T..G.AAAAA.T.A.CCT...AC..TTTAGA..GCT.AAC.A.GCT..C.GT.AATA.C.CCC.AAC..A.TT.CAAAC.A.GCC.AA.T.GAAA.GAAC..C.CGAT.G.A.CT.A..T...TA..C.
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) TTTTTTAATCATATTTTGTAGTTATAAGAAGTTGATAGAATATAAAGTGAGTGG-AATCTAGTGTTCCTTATGCGAGGAGGAGTGGAATTTGGTGCTAGGAGTTGCTGTTGCAAAGCAAAAGAGCAGTCATGGGTGCAGTCGGGGGCGAG
TuCypB-A(P) .....-......T.................A.......G...............-...T.G.......G....T..........................................................A....G............
Rice (P) C.C..G.TCG..T.A.CTGCA.C.CC.CTCCC.C..TT-.C.C...ACA.CCC.C....G.AA...A.A-----A.AA.AA.AA.C.CC.CAA..AGT.TGA..AA...G.AGTTAA.C.C..AG.TCTT..CT..-..A.A.AAC.CGA
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
AetCypB-D(P) CAGCTTGTGA-----GGCCGTGGTCTGACTGTTCGACCCCACCTGTCAGCCGAACGAGCAGCTCTGTTTATCTCGCCCGTGGACTCTCCCTCTCGGAGACCGCGTAGTGGAACGAGTAAACAGATCTGCTCTCTCCCTCTCTTTCTCTCT
TuCypB-A(P) ....C.....TGTGA......A.......A.C................A..A....GC......C........................T........G.T..............C.............C.....--------.......
Rice (P) ..--C..AC.TGTAG....C--AC...T.A.GCA.CG.ATCG..T...C..TCTT.GCT.CTAT.TC..CGTGGA.T.T...G.AGAGTG---..........AGT.....G.A.CAG.T.TATC.AT.CA..C.ATC...GA..CT..A
1210 1220 1230 1240
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .
AetCypB-D(P) CGTCCGCGGCGATACGATCCGAGATG---------------------
TuCypB-A(P) ....................A.....---------------------
Rice (P) TTC.T..CCG.CGG...G.GAGAGA.AGGCGGAGGAGATCCGAGATG
B
261
A: sequence from clones or directly PCR product with CypB gene sections in wheat species. B: sequence from clones with promoter sections of TuCypB-A in T. urartu and AetCypB-D in Ae. tauschii. Dots indicate bases identical to the top line; broken lines indicate gaps and incomplete amplification at 5’and 3’ends. Start codon (ATG) and stop codon (TGA) are shown in bold and underlined; HaeIII digested sites are showed in bold. The six introns are shaded.
262
Appendix III Nucleotide sequence of promoter, genomic DNA and cDNA of the putative rice CypB gene and its deduced amino acid sequence -1000 ERSE-II
AGAAGCTTAA TAACGAGCTG TACTTACTTA CCGTGAATTC CTATCATTGG AGTATGTGTG CACGTCATTA GTGCCATGCA
-920 Prolamin-box
TTGTTTGGAG CCTGTTTCTG CTGATTATAA TGTGACCTGC TTTGTGTAAT TGTTGTAAAA TGAGAGTGTT CTCTCAATAT
-840
TTTCCCATGG CAATTCAGAT TGGATATGCA ATGCAAGCTT ATATACATGC GAACACAATG TCATATTCCT CTTTGATTTA
-760
GATTATTGAG CGGATTATTC CATGAATTAT CAATTTGATT ATGACTATAA ATTAAATAAT TTATAAAAAT AAACTTAATA
-680
AATAGTTTAA AACACGCATT TTTAAGCCTT GTACAAGAAA ATTCTTTTTA TAAAAAAAAT TTAAAGCGTT GAGCAAATAA
-600
TCCGTTTCTA TAATTAGAAT AAGGTTTAGT TTGATTGTGA AGTTTGGATT TTTAATGGCA CGTAAAATAA GACTAGACTA
-520 TATA-box
ATTAAGTATT AATTATTTAA AAATTATTTG TTTTTTAAAT AAACTTCTAA ACAAAAAGTA TTTGGAAAAA AATAAACCTT
-440 Prolamin-box
TAACAGTTTA GAAAGCTAAA CTATGCTCAC AGTAAATAAC ACCCAAACTA ACTTTCAAAC TAAGCCTAAG TTGAAAAGAA
-360 AT-rich
CAACTCGATA GAATCTAAAA TTTTTATGCT CTCTTGATCG ATTTATCTGC ATCACCACTC CCTCATTTAC ACAAAACAAC
-280 CAAT-box CAAT-box
CCGCAATCGA AAGTTACAAA AAAAAAAAGC ACCTCAATGA GTGTGATTAA TGTGGAGTTA AACACGAAGA TCTTTGCTTG
-200 E-box
AGACAGAACC CGACACTGAC ATGTAGGGCC CACCTGTCAG GCAGCGCATC GCTTTCACCC TCTTGGCTAC TATTTCTTCG
-120 GC-rich CT-leader box
TGGACTCTTG GGCAGAGTGC GGAGACCGCA GTGTGGAGCA ACAGATCTAT CCATCCATCC CATCCTCGAT CCTTCATTCC
-40 S.C. ~~~~~~~~40
TGCCCGGCGG CGAGCGAGAG AGAGGCGGAG GAGATCCGAG ATGGCGGGGA GCGGGTGGAG GAGGATCCCG GCGGTGAGGA
~ ATGGCGGGGA GCGGGTGGAG GAGGATCCCG GCGGTGAGGA
M--A--G--S --G--W--R- -R--I--P-- A--V--R--R
120
GGCCGCCCAT GTCGCGCCCG GCCTCGGTCT GCCTCTGGAT CGTGCTCGTC GCCGCCACCC TGGCGCTCGC CCAGGTGCTC
GGCCGCCCAT GTCGCGCCCG GCCTCGGTCT GCCTCTGGAT CGTGCTCGTC GCCGCCACCC TGGCGCTCGC CCAG------
--P--P--M- -S--R--P-- A--S--V--C --L--W--I- -V--L--V-- A--A--T--L --A--L--A- -Q--------
200
ATCCACCTCG TCCGTCTCCA CTCTCCACCA CGCCTCGCTC CTCCTCCTGC TTCCCCGTAT CTGCGTGTGA TGTGATGCGT
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
280
GTGCTCTCCG ATCTGATTCG ATTGTTTGTG CTCGTGCGCT GATTTCTTGA TCTGTTTGAT CTGTGGATCC AATTGGCCCT
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
360
GGTGTGCTGA TTTGTTAGAA TTTTGAGTGG TGCCTCTATG TGCATCTATG CGTACTCACC TTTTTTTTTT TGTTGCTGTT
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
440
TAGGCGAAGA AATCGAAGGC GGATTTGACT GAGGTCACCC ACAAGGTCTA CTTCGACGTC GAGATTGATG GCAAGCCCGC
---GCGAAGA AATCGAAGGC GGATTTGACT GAGGTCACCC ACAAGGTCTA CTTCGACGTC GAGATTGATG GCAAGCCCGC
---A--K--K --S--K--A- -D--L--T-- E--V--T--H --K--V--Y- -F--D--V-- E--I--D--G --K--P--A-
520
AGGTTTGTGT TGGCCCCCTA TGTTTAGCTC TACTACCATA TCAGCATAAT ATATAAGTCT TCAGGCAGAA AGAAGAAAAG
AG-------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
-G-------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
600
ATATGCATAG TTTGATGTGT CTAGCATTAG CAAATACGGA GCAGTAACAA TTTAATTTGC ATTAATTGTG TCCCACCGTT
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
680
GTGTATAAGT GACTTGTGAT GGGCTGATGT CATATGCAGT GATTATTACA GTTAGAGTTA TCAGCATTTG AGATTGACCT
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
760
GAAGCTAACT CTATTATGAT GAGAATTTAG CTTCTTCTAT TGTTGTTTAT GTTTAAGGAG AAACGTTGAT GTGATATTTG
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
840
TGTATGTACT ATTTATAATG TCTTTCCTGT TAATGATTGC CTTACTGACA TTTATTATTT TTTCTTGTAT TGCCATAGGA
---------- ---------- ---------- ---------- ---------- ---------- ---------- --------GA
263
920
CGGGTTGTCA TGGGACTTTT CGGGAAGACT GTTCCTAAAA CGGCAGGTAC CATTTGCTTA TCATTTAGTT AGGTAACAGG
CGGGTTGTCA TGGGACTTTT CGGGAAGACT GTTCCTAAAA CGGCAG---- ---------- ---------- ----------
R--V--V--M --G--L--F- -G--K--T-- V--P--K--T --A--E---- ---------- ---------- ----------
1000
AAATTCATTG TCATATCACA AATGCTAATG ATTCACTATA TCTTTTTTTT TTCCTACAGA GAACTTCCGA GCACTTTGCA
---------- ---------- ---------- ---------- ---------- ---------A GAACTTCCGA GCACTTTGCA
---------- ---------- ---------- ---------- ---------- ---------- -N--F--R-- A--L--C--T
1080
CAGGTATGCT CCCTGATGAT TTATACTGTT TTTATTACAT TAGATGTGTT CATTCATCTT CTGCTAGAGT TTCCAGATGG
CAG------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
--G------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
1160
TTAATCCCAT GGCAAGCTTG GTAGTGGCAG CATGTAGAAT TTCCAGCATT GAATAACACT TTCTTTGCCA CTTTAACCTG
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
1240
AAATTATTTT TGAATGCACA ACTTTTGTGT CATTATCAAG TTTGGCCAAT CAAATATACT GTTACAGTTG ACTAGTTAAA
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
1320
AATTGATGCA AAAATACATC AGCTCAGCTG TTGTCATTAT TCATACGCAA GTGTAGAATA TTATGAAAAC CCATAAAAGT
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
1400
ATTTACTTCA TATAATGAGA GAGATCCCAT TCTGGACAGC GAATAGCACT ACAATCTGTG TGGTAGCATT CCCTGATGCC
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
---------- ---------- ---------- ---------- ---------- ---------- ---------- ----------
1480
TACTTGTGGT TATTATACGT AATCTAAGTT TGGGTTTGAT GTAGCGCTTT CAACTTTGTT GTTATACAGG AGAGAAAGGA
---------- ---------- ---------- ---------- ---------- ---------- ---------G AGAGAAAGGA
---------- ---------- ---------- ---------- ---------- ---------- ---------- -E--K--G--
1560
ACTGGAAAGA GTGGCAAAGC ACTCCACTTC AAGGGAAGTG CATTCCACAG AATTATACCC AGCTTTATGA TCCAAGGAGG
ACTGGAAAGA GTGGCAAAGC ACTCCACTTC AAGGGAAGTG CATTCCACAG AATTATACCC AGCTTTATGA TCCAAGGAGG
T--G--K--S --G--K--A- -L--H--F-- K--G--S--A --F--H--R- -I--I--P-- S--F--M--I --Q--G--G-
1640
TGACTTCACA CTTGGTGATG GAAGGGGTGG TGAATCTATC TATGGGACGA AGTTCGCCGA TGAAAACTTC AAGATCAAGC
-D--F--T-- L--G--D--G --R--G--G- -E--S--I-- Y--G--T--K --F--A--D- -E--N--F-- K--I--K--H
1720
ACACCGGACC AGGTAATTTT CGTTATTTCC TGAACCATTT TTTTACTACA CTATAGACGC ATTATTTGTG TGGTAGACCA
ACACCGGACC AG-------- ---------- ---------- ---------- ---------- ---------- ----------
--T--G--P- -G-------- ---------- ---------- ---------- ---------- ---------- ----------
1800
TCAAATCATC CTTGTTTGAT TTGATCTCAT GTTTATGCAG GCCTCCTGTC CATGGCCAAT GCTGGGAGAG ACACAAACGG
---------- ---------- ---------- ---------- GCCTCCTGTC CATGGCCAAT GCTGGGAGAG ACACAAACGG
---------- ---------- ---------- ---------- --L--L--S- -M--A--N-- A--G--R--D --T--N--G-
1880
GTCCCAGTTT TTCATCACCA CTGTAACCAC CAGCTGGTAA TCGTCGTTTC CTCACTAGCC TTTGTAATCA TAGCAGCACT
GTCCCAGTTT TTCATCACCA CTGTAACCAC CAGCTG---- ---------- ---------- ---------- ----------
-S--Q--F-- F--I--T--T --V--T--T- -S--W----- ---------- ---------- ---------- ----------
1960
ATTTCTAATT TGTACTGAAG ACACTTCAAT CTGTTGAAAC TATTTATTTG ATTAGGTTGG ACGGGAAGCA CGTCGTGTTC
---------- ---------- ---------- ---------- ---------- -----GTTGG ACGGGAAGCA CGTCGTGTTC
---------- ---------- ---------- ---------- ---------- ------L--D --G--K--H- -V--V--F--
2040
GGTAAGGTGC TGTCTGGAAT GGATGTGGTT TACAAGATTG AAGCTGAGGG CCAGCAGAGT GGGTCACCGA AGAGCAAAGT
GGTAAGGTGC TGTCTGGAAT GGATGTGGTT TACAAGATTG AAGCTGAGGG CCAGCAGAGT GGGTCACCGA AGAGCAAAGT
G--K--V--L --S--G--M- -D--V--V-- Y--K--I--E --A--E--G- -Q--Q--S-- G--S--P--K --S--K--V-
2074
TGTCATCGCG GACAGCGGCG AACTGCCGAT GTAA
TGTCATCGCG GACAGCGGCG AACTGCCGAT GTAA
-V--I--A-- D--S--G--E --L--P--M- -* The minus numbers on the left side indicate position from start codon (S.C.) in the promoter sequence. All the regulated elements identified are bold and underlined; and names of the elements are presented above the element motifs. The putative CypB genomic sequence is presented in the first line with the numbers indicating the genomic base pair from start codon. Second line presents the CypB cDNA and the third line presents its amino acid sequence. Broken lines indicate the introns of CypB genomic sequence at the top line; *indicates stop codon of the translation.
264
Appendix IV Blots obtained from GrainGenes for probes matching wheat Cyps and PDILs
265
266
267
268
Appendix V DNA alignments showing the alternative splice forms encoding rice Cyps and PDILs
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 CTCTTGGCTACTATTTCTTCGTGGACTCTTGGGCAGAGTGCGGAGACCGCAGTGTGGAGCAACAGATCTATCCATCCATCCCATCCTCGATCCTTCATTCCTGCCCGGCGGCGAGCGAGAGAGAGGCGGAGGAGATCCGAGATGGCGGGG
OsCYP24-2 ---------------------------------------------------------------------------------------------------------------------------------------------.........
OsCYP24-1 ---------------------------------------------------------------------------------------------------------------------------------------------.........
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 AGCGGGTGGAGGAGGATCCCGGCGGTGAGGAGGCCGCCCATGTCGCGCCCGGCCTCGGTCTGCCTCTGGATCGTGCTCGTCGCCGCCACCCTGGCGCTCGCCCAGGTGCTCATCCACCTCGTCCGTCTCCACTCTCCACCACGCCTCGCT
OsCYP24-2 ........................................................................................................................------------------------------
OsCYP24-1 .........................................................................................................---------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 CCTCCTCCTGCTTCCCCGTATCTGCGTGTGATGTGATGCGTGTGCTCTCCGATCTGATTCGATTGTTTGTGCTCGTGCGCTGATTTCTTGATCTGTTTGATCTGTGGATCCAATTGGCCCTGGTGTGCTGATTTGTTAGAATTTTGAGTG
OsCYP24-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP24-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 GTGCCTCTATGTGCATCTATGCGTACTCACCTTTTTTTTTTTGTTGCTGTTTAGGCGAAGAAATCGAAGGCGGATTTGACTGAGGTCACCCACAAGGTCTACTTCGACGTCGAGATTGATGGCAAGCCCGCAGGTTTGTGTTGGCCCCCT
OsCYP24-2 ------------------------------------------------------................................................................................----------------
OsCYP24-1 ------------------------------------------------------................................................................................----------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 ATGTTTAGCTCTACTACCATATCAGCATAATATATAAGTCTTCAGGCAGAAAGAAGAAAAGATATGCATAGTTTGATGTGTCTAGCATTAGCAAATACGGAGCAGTAACAATTTAATTTGCATTAATTGTGTCCCACCGTTGTGTATAAG
OsCYP24-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP24-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 TGACTTGTGATGGGCTGATGTCATATGCAGTGATTATTACAGTTAGAGTTATCAGCATTTGAGATTGACCTGAAGCTAACTCTATTATGATGAGAATTTAGCTTCTTCTATTGTTGTTTATGTTTAAGGAGAAACGTTGATGTGATATTT
OsCYP24-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP24-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 GTGTATGTACTATTTATAATGTCTTTCCTGTTAATGATTGCCTTACTGACATTTATTATTTTTTCTTGTATTGCCATAGGACGGGTTGTCATGGGACTTTTCGGGAAGACTGTTCCTAAAACGGCAGGTACCATTTGCTTATCATTTAGT
OsCYP24-2 --------------------------------------------------------------------------------...............................................-----------------------
OsCYP24-1 --------------------------------------------------------------------------------...............................................-----------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 TAGGTAACAGGAAATTCATTGTCATATCACAAATGCTAATGATTCACTATATCTTTTTTTTTTCCTACAGAGAACTTCCGAGCACTTTGCACAGGTATGCTCCCTGATGATTTATACTGTTTTTATTACATTAGATGTGTTCATTCATCT
OsCYP24-2 ----------------------------------------------------------------------........................--------------------------------------------------------
OsCYP24-1 ----------------------------------------------------------------------........................--------------------------------------------------------
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 TCTGCTAGAGTTTCCAGATGGTTAATCCCATGGCAAGCTTGGTAGTGGCAGCATGTAGAATTTCCAGCATTGAATAACACTTTCTTTGCCACTTTAACCTGAAATTATTTTTGAATGCACAACTTTTGTGTCATTATCAAGTTTGGCCAA
OsCYP24-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP24-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
AExon I
ExonI
Intron IIntron I
Exon II
Exon II
Intron II
Intron II
Exon III
Exon III
Intron III
Intron III
Exon IV
Exon IV
Intron IV
Intron VI
269
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 TCAAATATACTGTTACAGTTGACTAGTTAAAAATTGATGCAAAAATACATCAGCTCAGCTGTTGTCATTATTCATACGCAAGTGTAGAATATTATGAAAACCCATAAAAGTATTTACTTCATATAATGAGAGAGATCCCATTCTGGACAG
OsCYP24-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP24-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 CGAATAGCACTACAATCTGTGTGGTAGCATTCCCTGATGCCTACTTGTGGTTATTATACGTAATCTAAGTTTGGGTTTGATGTAGCGCTTTCAACTTTGTTGTTATACAGGAGAGAAAGGAACTGGAAAGAGTGGCAAAGCACTCCACTT
OsCYP24-2 --------------------------------------------------------------------------------------------------------------........................................
OsCYP24-1 --------------------------------------------------------------------------------------------------------------........................................
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 CAAGGGAAGTGCATTCCACAGAATTATACCCAGCTTTATGATCCAAGGAGGTGACTTCACACTTGGTGATGGAAGGGGTGGTGAATCTATCTATGGGACGAAGTTCGCCGATGAAAACTTCAAGATCAAGCACACCGGACCAGGTAATTT
OsCYP24-2 ...............................................................................................................................................-------
OsCYP24-1 ...............................................................................................................................................-------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 TCGTTATTTCCTGAACCATTTTTTTACTACACTATAGACGCATTATTTGTGTGGTAGACCATCAAATCATCCTTGTTTGATTTGATCTCATGTTTATGCAGGCCTCCTGTCCATGGCCAATGCTGGGAGAGACACAAACGGGTCCCAGTT
OsCYP24-2 -----------------------------------------------------------------------------------------------------.................................................
OsCYP24-1 -----------------------------------------------------------------------------------------------------.................................................
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 TTTCATCACCACTGTAACCACCAGCTGGTAATCGTCGTTTCCTCACTAGCCTTTGTAATCATAGCAGCACTATTTCTAATTTGTACTGAAGACACTTCAATCTGTTGAAACTATTTATTTGATTAGGTTGGACGGGAAGCACGTCGTGTT
OsCYP24-2 .............................---------------------------------------------------------------------------------------------------......................
OsCYP24-1 .............................---------------------------------------------------------------------------------------------------......................
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 CGGTAAGGTGCTGTCTGGAATGGATGTGGTTTACAAGATTGAAGCTGAGGGCCAGCAGAGTGGGTCACCGAAGAGCAAAGTTGTCATCGCGGACAGCGGCGAACTGCCGATGTAATGAGCTGAAATGATGTTTCACTGAAGCTACTGCTA
OsCYP24-2 ...................................................................................................................-----------------------------------
OsCYP24-1 ...................................................................................................................-----------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 CTGCTTCTCGTTGTAGAACTGTTATTAGCTGCTCTGTTTTGCTATGCTTCTACCCCGAATCTGGTTGGAGTAAATTTCTGTTAGACTATAACAGCGTGCTTATATCGCCTCTTAGTTGTGGGCGATTTGGAGTGAACTAGCATAAAAAGG
OsCYP24-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP24-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
06g49480 TCGAAACTTTACTTAAGTCGTCTTCTTGTTCTAAGTCGAATTATACTTTGAAGTGACTTGCTTACTGTTGAGCGCTTG------------------------------------------------------------------------
OsCYP24-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP24-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon V
Exon V
Intron V
Intron V
Exon VI
Exon VI
Intron VI
Intron VI
Exon VII
Exon VII
270
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 AGCTCACTGGCTGGGGATAGACGAGACGGCGACCACGACGATGACGATGACGACGACGACGGCGACGGCAGCAACGGCATCCCCACCACCACCCCCATCTCCGGTTCCTCCCCTCCCCGCGTCTCAACCCACCGGAAATCCATTCAAAAG
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 CACCCGCGCTCCCGCACCAATCCACCGCACGCCATGGCGTCGCGAGCGGCGGCCGCGATGGTGCTCGCCGGTGCCCTTCCTGTGTTTCCCGGCCGTCCCCTGGTCGCAGCCGGCGCCAGGTGCTGCGACGGCGGCATTCGCGGGAGGGTC
OsCYP50 ---------------------------------.....................................................................................................................
OsCYP46 ---------------------------------.....................................................................................................................
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TCCTGCTCCTCCCACCGTCGCTCGGACCATCCCTCCTGCGCGGCTGAGGTGAGCACGCGCTGGTTGCTGGTCTCTCCTCATCCAAATGGCTTCCAAAGCTGTAGCGTTCGCTTCAGTTTAATCGTGTGGTTGTCTGGTTTGGTGCAGGAG
OsCYP50 ......................................................................................................................................................
OsCYP46 ................................................---------------------------------------------------------------------------------------------------...
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GGAGGAGTGGTGGAGCTGCTCAAGGGCGCGGTCGCGGCGCTCGCCGTCATCGCCCAGATTTCCGTGTCGCTCCCGGCGGACGCGATCCTCTACTCGCCGGACACGAACGTGCCGAGGACGGGGGAGCTGGCGCTGAGGAGGGCCATCCCC
OsCYP50 ......................................................................................................................................................
OsCYP46 ......................................................................................................................................................
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GCGAACCCAAACATGAAGACCATACAGGTAAACTGCATCAGAATCGGCGGCGATGCATATCGCTTAGTACGGTTGGCTGGGCGTTGTTCTTAGATTGTGCGAGCCCATTTGCCAGGAATCGCTGGAGGACATTTCGTATTTGCTGAGGAT
OsCYP50 ...........................----------------------------------------------------------------------------------------...................................
OsCYP46 ...........................----------------------------------------------------------------------------------------...................................
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 ACCGCAGAGGAAGCCGTATGGCTCCATGGAAGGAGACGTAAAAAAGGCCATGAAGGTAATAAGTTGACTCTGTGTCTTCTTCATAAGCATTTTTCTAGCAACAATGTTTTGCTTCTTCTATGCATTGTGAACTTCTTATTGAACTTGCTG
OsCYP50 .......................................................-----------------------------------------------------------------------------------------------
OsCYP46 .......................................................-----------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 AGCTGTTGTCCTGAATGCTGATATATAGAACAAATGCGCTCTGGATGCAGATAGCAATGGACAACAAGGATGCGATCTTGGCAAGTATACCTGTAGAACTCAAGGAAAAGGGCTCGAAGTTATATACCTCCTTGCTTGAAGAGAAGGTCA
OsCYP50 --------------------------------------------------................................................................................................----
OsCYP46 --------------------------------------------------................................................................................................----
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GTAGGTTCTAGGATGTGCTCATGGTGAATTGCAGTTTATGAAACAGATCAAATTCTGGTTTTCACTTGTACTTATTTATAAATTATACCTGAGCTACTTTGGAATCAACTGCAGGGTGGTTTGCAAACTCTCTTGAAATATATAAAGGAA
OsCYP50 ------------------------------------------------------------------------------------------------------------------....................................
OsCYP46 ------------------------------------------------------------------------------------------------------------------....................................
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 AATGATCCTGACAGACTTTCGGTTGCACTTGCTTCTTCTCTTGATACTGTTGCTGAATTGGAGCTGTTACAGGTAACTGTACTAAACTGAATAGAAGTTTCATTATGTTTAAAGGAGGATGATTATACTCCGGCGATGATCGGATTACTT
OsCYP50 ........................................................................------------------------------------------------------------------------------
OsCYP46 ........................................................................------------------------------------------------------------------------------
B
Exon I
Exon I
Intron I Exon II
Intron I
Intron II
Exon II
Exon III
Intron II
Intron III
Exon III
Exon IV
Intron III
Intron V
Exon IV
Exon V
Intron IV
Intron V
271
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 AAAGGGGGAAATTTCTTTCAACAGGCTCCAGGTTTATCCTTCCTCTTACCACAACAATATTTGGAATATCCAAGGTAACTTTTGTTGATCTCTCCAAAGCTTAAAATCTAAATGTTTCTTTGTCTCTAGAATTATTGTGAGGGAAGGGCA
OsCYP50 ------------------------..................................................----------------------------------------------------------------------------
OsCYP46 ------------------------..................................................----------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GAGATAGGTCCGAAGATTGAAGGTGACATTTGATTATACTGAGTTACTGACATGCATGGTTCTATGTCCTGCAAACAAGTATAAGCTCTATGTCAGTATAAGTTTTAAGATAACCAGGTCTCTGCCCCCAAGTTCTTGATGGTAATAATC
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 AAAGCTGCCGCTATTAGTAGGTTTTCAAAATGAATTCAATAGAAAACTTACCAGTTTTTCCATTGTTGCAAATTAATTGGTTTTGACAAACTTGCCATTTTATCATATTTGCAAACTCACTATTTATTGCTGGTGTCACCCTTCATATAT
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TTCATGGAGTAGAAACTGTCGAATAAAAAGTACTTTCTAGTTAATTCCAAGTGGGAACTTATTTTTATTTATAACATATATCATCACATAGTATTATAAAATCATTAAGGCCAGATTGGTACAGGGTGGCAAGAAAACCTCACAAGCATT
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TTTCTTTTCGAACTCTTGAGGAATGGTGAATTTTTCTTGGGTTCTGATTGGCTGATCGCTTCAGCAGCAATGTCGGTAAATTTGAGCGCATGTTTTTAGTTTACTTAGTCGTCCTTTAGGTCCCCTGTTTTTCTTCTGTTTTGGGTCTTT
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GGGGTCGGTGAAGTATGGGCCCAGGTTGTTGTCCAAGTTGTCCATTTTAGGTGCGCACTTTTGCGCCAGAGCGAGTCGTGTCCTTGTCGCGCCCGGCACATTCTGATCATGTGGGATGGCACACGACACGGTGGAACGTGTCATGTGCAT
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GAAGGCCTCAATGGAAACTACTTTGGGCCTCCTGTAATCCGTATAAACAACAGTGGAGAAAACTGAGAAAAGCTACAGTGAGATTGCGCAGCGCCAAATTCATTCGCGTCTCTGTTCTTTGTTCTTGTGAGTGTTCCTTCAATCCCAGGG
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GGATCGCTGACAAGCAATACACTCAGTATGTGTAGCTGATTAGCAGATTATGTAAGGTGGTGTTTGGATGGGAGGGGGACTAAAATAAGTCCCTCCCATCCCAAACAGGCCCTAAGTGTAATGGTGCTTTGAACATTCCATCCCTGTGGC
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 ACTTATATTAAGCATGCAATCATTCCTCCAGTCTGTAACGAAATGTTCATTTGTACATTTAATTTAAACAGCTCGCTTTTAATAGGCTAGCAGGAAGAGGAGTTGTTGAATTCTCAGTGGAGAAAGGTGATGGTTCAACATTCTTTCCAA
OsCYP50 -------------------------------------------------------------------------------------.................................................................
OsCYP46 -------------------------------------------------------------------------------------.................................................................
Exon V
Exon VI
Intron V
Intron VI
Exon VI
Exon VII
272
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 CTGCTGGTGGTGAACCAAAAAGTGTTGCTACAATTCAGGTGGCTTTGAATGGCTGTGTAATTGTCTGGTATCATAAACTGCAGTCTTTTGACATTGAGAAATAAGAACCTTATCCTTGGTTGTTATCTTTTGTCATACCATGGTGTGCAA
OsCYP50 ......................................----------------------------------------------------------------------------------------------------------------
OsCYP46 ......................................----------------------------------------------------------------------------------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GAGTATGGTTCTGTATACAGTTGCAGTTATAAAATTCCACGTGTCAGCTATTGATTTCTTTACTAATTTCCAGGTAGTGATTGATGGATATTCTGCCCCACTGACTGCTGGAAACTTTGCGAAATTGGTAAGGTAGCTTAACTCCAACCT
OsCYP50 -------------------------------------------------------------------------......................................................-----------------------
OsCYP46 -------------------------------------------------------------------------......................................................-----------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 CATCTCACTATTGATTATTTCCTGCCAATAGCTCTGCCTTCTGACATATATGATAAAGATCATCGAAATGATTGTGTTCATCGACATAACATCTTCGAGAACATCAATTTAAGATTTCATGTTTCTGCGATGTGCTAAAAAAATTTCCTT
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TACTAGCTCTTGATTTTCTTTTCAGTAGTTAGGTTACATCATCTGATCCAATGAAATGAACTTTTCTTCAGGTGTTGGATGGGGCATATGATGGGATAAAACTGAAATGTGCAAGTCAGGCAATTATTGCAGACAACGAGAATGGAAAGA
OsCYP50 -----------------------------------------------------------------------...............................................................................
OsCYP46 -----------------------------------------------------------------------...............................................................................
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 AAGGTTACACTGTTCCATTAGAGGTCATGCCTGCTGGGCAGTTTGAACCGTTATACAGAACTCCGCTAAGTATTCAGGTCAGTATTCCACACTGTTAGGTCTGTTCTGAAGGGGTATTTGGATCTAAATCATCAATCAAAATCCTTACGA
OsCYP50 .............................................................................-------------------------------------------------------------------------
OsCYP46 .............................................................................-------------------------------------------------------------------------
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TTTAGTTCAAACTGGAAGTGTATGTAAAAAGGATATTAAAGAAAGCCCACTCATCTGGACTACCCTGTAAATGCCAGTGAAATCTGAGAGTTACCGTCTTTCGTAAGTCATAACCCAGTTCCTTTGTTACTTGATCCAATACAGGATGGA
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------......
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------......
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GAATTGCCAGTCCTTCCGATGTCTGTATATGGTGCCGTTGCTATGGCCCATAGTGTGGATTCTGATGAATACTCGTCACCAAGCCAATTCTTCTTCTATCTGTATGACAAGAGAAATGTGAGTAAATGGATATATCTTTAGAACTAGACA
OsCYP50 .....................................................................................................................---------------------------------
OsCYP46 .....................................................................................................................---------------------------------
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TATTCGCTAGCACAGTATTTTAAATTCAGATTGCATAAGTGCATGTACCTCAACAGTGCACCCGTCTACCTCTTGTGTTGTGAACCATATTTCTGAATGCTTCTGCTTTTCTAGACCCATAGCGTGCCCTATGTATAAGAACTAATTGCG
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TAAATATTTTAATTTGTGATTATTTTCCTTTATCTTGTGCAGTCTGGTTTAGGAGGAATATCTTTTGATGAAGGGCAATTCTCAGTATTCGGGTAAGTAGTGATTGCCATTACAGAACTGTGACCACTAGACAGCAAACTTGGTTTATTT
OsCYP50 ------------------------------------------....................................................--------------------------------------------------------
OsCYP46 ------------------------------------------....................................................--------------------------------------------------------
Intron VI
Intron VII
Exon VII
Exon VIII
Intron VII
Intron VIII
Exon VIII
Exon IX
Intron VIII
Intron IX
Exon IX
Exon X
Intron IX
Intron X
Exon X
Exon XI
Intron X
Intron XI
273
4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TGTGGATTAACAACAGAGAACTGTGTCGATCAAACATCTGCAGGTATACCACAGATGGAAGAGAAGTTCTGTCGCAGATTAAAACTGGAGATATAATTCGTTCTGCCAAGCTTGTACAAGGTAGAGAGCGCCTTGTACTGCCTCCAGAAG
OsCYP50 ---------------------------------------------.........................................................................................................
OsCYP46 ---------------------------------------------.........................................................................................................
4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 CGCCGGCAGAGAGCTGATACAACGGCAAGCTTCGCAAACTGGAAACATTCTTTCATTGGTCAGGAGACTTAGCATATTTTATACCATAAATCATGTAAACCTGACTGAGCAGTTCATCAAATTTATATATAGAATCACTGCAAATGTTCA
OsCYP50 .................-------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 .................-------------------------------------------------------------------------------------------------------------------------------------
4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TCAAATTTATATATAGAATCACTGCAAATTTGCACATTTAATCATTCTTTTTGTGCATTTGCTACTTCACAATAATCTGAATAGAGTGGAGTGAATCCTGGATACTAATCTTGTATTGTCAGGAAGCTGCCCTCCTCCCTCGGCGTGCAT
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 CTCTCGTTGAGCCACGCGAGAATGTCAGCGCGGACGCGCTCGATGTTCTCGTCAGGCTCCCCGAAGAGCATGGAGTGCATCATCCCGTCGTATATCTTGATGGTCTTGTCCTTGCTGGCGGCGGCGTCGTACAGCGCGCGGCTGATGTCC
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
4660 4670 4680 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GGGTCGGTCACCTCGTCGGCGCTGCCGTGCACGACGAGGAACGGGACGGTGACTTCGCCGAGGCGGGCGCCGAGCTCGTCGGTGGCGCGCAGCAGCTCGACGACGGTGCCGAGCCTCGGCCGGCCGCTATAGCGCATGGGGTTGCGCGCG
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
4810 4820 4830 4840 4850 4860 4870 4880 4890 4900 4910 4920 4930 4940 4950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GCGATGAGGCGCTTGGCCGGCACCTTGACGGACTTCTCGATGAGGTCGGCGGTGGGGACGATGGCGAGCGTGGGCGCGAAGCGGGCGACGAAGGTGAGGATCTGCGGCAGCGGCCATGGCGGGCGGATCCGGTCGGAGATCTTGCACATG
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
4960 4970 4980 4990 5000 5010 5020 5030 5040 5050 5060 5070 5080 5090 5100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GGCGCCACCAGCACGGCGCCGGCCCACTCCTCCGGCGGCGTGCGGAGGTGGATGAGGAGGCAGATGGCCCCGCCCATGGACTCCCCGAACAGGAAGCACGGCAGCCCGGCGTGCTCCTCCCGCCGCCGGACGGAGCGGAAGAAGGCGAGC
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
5110 5120 5130 5140 5150 5160 5170 5180 5190 5200 5210 5220 5230 5240 5250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 AGGTCGGCGATGGCGGAATCGAGGTCGGGGACGAACGCGCGGAGGCCGTGGGAGCGGCCATGGCCGGGGAGGTCGGCGGCGAAGCAGGCGAAGCCGGAGCGGGCGAGGAAGACGGCCGTGGACTGGAACGTCCAGCTGATGTCGTTGCCG
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
5260 5270 5280 5290 5300 5310 5320 5330 5340 5350 5360 5370 5380 5390 5400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 TAGCCGTGCACCATGAACACGAGCGCCCGCGGCGCGCCGTCGCCACGGGGCCGCCACGCCCGCGTGAAGAGGCGGCGCCCGCCCGGCGGGGTGAAGTAGGACGACTCGCCATCCGCGCCGTGCGCCGCGTAGTACTCAGCCTCCGTCGCC
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon XI
Exon XII
274
5410 5420 5430 5440 5450 5460 5470 5480 5490 5500 5510 5520 5530 5540 5550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
07g37830 GGCTGCTCGCCCCAGAAGTTCACATCCGGGGCCTCCTCATGCCGCTCGCCGTCCGGCATGGTGGGGAATCGGTTGGGCCGGTGCGTGGCGTGCGGATGGGGAACAGGTGGTGGTGGTGGTGGCCGGAGAGTTGGGGAGTCGTTGCGTAGC
OsCYP50 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP46 ------------------------------------------------------------------------------------------------------------------------------------------------------
5560 5570 5580 5590
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . .
07g37830 AGCAGGGGAGTTTACGATTCGTTTCGTTCGGCGTTTCGGTTTT
OsCYP50 -------------------------------------------
OsCYP46 -------------------------------------------
275
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 AGCCGGCACAATTCCCATGTAAAAGGGATATTGAGATAAGGGCGGAGTAATCGAGTATCGCGTAAAAGAAGAAATCTCCCGTCTCGTTCTCGTTCTCCTCCGCTCCTTGCGCCGCCGCCAAGACGAGCGGAGGGCCGGGCGGCGATCTCC
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 ATCTGGCAAGCAGAGCAGCGGAGGGGAGGAGGGGATCCTGGTGCGTCCTCTTTCTGATCCATCTCTCTCTCTCTCTCTCTCTCTCTCTCCCACCCCACCGGGACCTGGAATTTGCTTGCGTTCGTGAATTTGATAGCCTTAAACCTCTCT
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TATTTCAAGTAATAACAGTTTGTTTGTTTACCTAAAAGTTCGGGGGTTCAACCGAACCCCCATGCCCCAAGTTGGATCCGCCCCTGGTTTGAATTGCTGAAACATAATTTCGAGCTAGTTAAACGAAGAGACAACATGCCTGGAAATGTA
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 AAAAAAAAGTATGCTGATTTGAATTTGATTGTTGGAACTGCCCTATAGGAAACTGGTAAATTTTAGAACAAAAAACCTTGCTTATAGTAAAATCGTGAATGGATGATATGCAAAGACATGGTGAGAAATATTCACTCGCTCATACTACAA
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CTACTTAAGAGGACCATATTTGCTCACTCTGGAATAGCTGCTGACCTGCTGTGACCATCATACACCCCCTCACCGCAATGCAGGTTCCATTTGAAGTAATCGGAGAATGGTATGTTGAGAATTAGATTAAAGCTTCCATTTTAAAATTGT
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 AATTGTTTGGGCTGTCAGCTATTTGCCATTATTCACAAGTGATAATTGTGACCCTACAACGAAGAGGCTAACAGTAGGTTTTAGAATTTCAGTAATGTTATATCTCAAAAGCCAACTATTACATTTGCAGGGAAGGATATTAGTGATTTT
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CTACTGCTGTTTCGTCAGAAGGACAAGAACTGAAGAATTCAGTTAGCAACAGTATGGTGTTGCCTTCATCAAATACCAGGGCCACTTCTTCCATGGCTTACCCTGTTTCCCTGAGCAGCCTCCACCACCACCCCAACAACCACCATGCTT
OsCYP40 -----------------------------------------------------.................................................................................................
OsCYP28 -----------------------------------------------------.................................................................................................
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TTTTTCTTCCCTCGAAAACAAACCATGATAATACTCATAAACCTATTGAATCCTCTAGAATTTCTAGACGGTCTCTCATCTTCCTCCCTGTCCTCCCTTCTCTTCTATACGCTTCATCATCCCCTGCCTTAGATGACGCAAACATTCCAT
OsCYP40 ......................................................................................................................................................
OsCYP28 ......................................................................................................................................................
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CCACCTCTGCCATTGATACCACCATCACAGATCGGATCTTCATGGACTTCAGTGTCTGCCCTAGCTACTTTCGCTCTGACCGCACTTTAGGAGCTGAGCTTGCTACATGTCCTGATTCTGAACCTCTTGGTCGTGTAATTTTTGGTTTGT
OsCYP40 ......................................................................................................................................................
OsCYP28 ......................................................................................................................................................
C
Exon I
Exon I
276
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 ATGGTCGACTCCTCCCCCTCACTACTGCCAACTTCAAAGCTGCTTGCACTTCTGCTGCATACCGGGGAACACTTGTCCACAAGCTCCTTCAGGGCCAGTTCTTCGTTGCTGGCCGCCAAGGTCCTCGGCGTGACAGGGGCGAGGTCCAAC
OsCYP40 ......................................................................................................................................................
OsCYP28 ......................................................................................................................................................
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CTCCCACCGGACTTGTGAGGAATGCTGAGACTATTGACCCTAAAGCATTTGAACTAAAGCATGCAAGGCCTGGCACCCTCTCATTGTGCCTTGGACAGAACGATGACGATGATGACATCAAGCTCAATCCTAATTATCACAATGTTGAAT
OsCYP40 ......................................................................................................................................................
OsCYP28 ......................................................................................................................................................
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TCTTGGTTACCACTGGACCAGGGCCCTGCCCAGAGCTTGATGGACAGAACATTGTCTTTGGAACTGTATTGGAAGGTTAGTTGCTTTCATTTTCTGAACTTTTACTAAATGGTTCTTGCACACTTTGTGTTAAATTCTGTCTTGCAAGAA
OsCYP40 ...........................................................................---------------------------------------------------------------------------
OsCYP28 ................................................................................----------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TTTCAATGTCTTTCCCTCGAACAAGTTGGGATTTGACCTGTGCGTAATCTACTAATGCAAGGTTGGCATGATTAAATTCATGCATAAATCTATTTTCTTTGGTTCAAACAGAGAAAAATAAAATGTATTTCAAACCGTTTCATTTTTTGT
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CAAGTATAATTATATGACATCTTCCATTCTCCTTTTGGTATAGTTATATACACTTGCTAAGGTAAACATGTTTTCATTGAAGTAATCAACATTAGTACATAACAGAGAAGAAAAAAAATGCAAATTTTGCAGCTTTGCATGAGTATTAAC
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 AAAATAACAAACAGTTGTGTCCGTATTCTGGCTAGGACTTCATCCATGCAGAACATAAAGTTAGATGTTTTATTATTACCAATTTCTGCAAGTAATATAAGCTTGTAAAGAAATTGTCATTTTATTTCTTCCTTTTTGTTGTCTCAACAC
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CAAGGATGGTTATCACACTAAGATTAATTCTTTTTTTTTTCTTCTCTGCACTTCACCACAGTATAACTCATCATCAATTCCTGCCATGGTTTGTTCTAACAATGTGCCACATTGTCAATAAACAACACCAGAGAGTGTATCATCCTCATA
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TAGTCAAATTTGGTGCATACTTCATTGTTTTTCCCTGTTCTGATTTATTAGATCTCTGGTGGTTATCAGAAGTAGATTGAACTAAACCAAGCAACTCATCAATGAGATTTATACTGCCACTGAACATGTCACCCCAAAAGAGAATCTACT
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TCTAGCTAATTTAAGACTGACATGTCTGCTTTGACAGTTTGATTTGTAATTTAATAAACTTTACAACAACAACCAGGGCAGTAGTTAACTACAAAGCAGTTCATATTTAAGTTTTATGCATCAAGAACTTAAAAGCACGGCCCTTCTATC
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
Intron I
277
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CCCAAAAAAGCACATGCCCACGTTCTGCATCTGCATGCCCATGCGGCATGCTCTCATGCACATGCATGGGCAGACGCAACAAATGCATGCGCACATACAACTATATAACTTATATTCACATGACACTTGAGGCACACTAATGCTTGTCAT
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TAGGACCCATTGCCACTTGAAGTGTAATAGTGTTACTCATTTGAACTGAACTAGCTACTTAGTCTCAGTTTCAGTATTTGATTATTACCAACAGTTTTTTCTTAAGCATCCATTTTTCTTCTTCTATAAGCAACTTTGATTGTCAAGTTC
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TGCATTCACATGCACACATAGGCGACTAAAACAGACTTTCAAGCACTGAGTTAAACAAGTGTATTAATTGGATATATTCCAGTGGAATTGACATGAAGCTTCAATTTTGTACGAGTTGCTTTCTGTTGTCTTAATGGAAACTAGTATTGC
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CACTGTATGATTAATGATGATATTGTTGATTCAGATTAACAGAAAGCGGCTAATTCGAAAGATTTTACATGATAATTTGTACATATGTAATTTGTAACTATTATATAGTTTTAATATATCGCAGTAGCAGTCTCTCCTGCTGTTTTCATC
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 GTCAAAAAAAAAAAAAACTGTTTTCTCCGCAATTCCTTTTTTTAACTATTTGGTCTTGCAACTTGTGTTTTTATCAGATTATTTCTGCACGATTGGCAGTCCATTCAATTCAAGGTTGAAATTTTTTACAGAAGAGCTAATGTGTTAATA
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 GTATAAATCAAGTGGATACTTCTAGCATAATAGAGAACTGTCTTGATTTCCTTGCCCAAAAAAAAAGAGGACCGAGTGGTTTTTAATCTATTTCTGATTAAGTCAGTGTATGAAAAAAAAAATCAGCTACCACTTATCTTTCTTTCATGA
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 AGAACCCCCTTTTTCTTAACAAAGCATACTCTCCGTATAGCCATGGAAGTATATATTAATTACATAAAACAGAAAGACTGTATGACATTTTTTTTGTGAATATGTAAAGGTAGCCATTACTATCTTTTTATTAGCCTCATTTTTCTTGAG
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CAAATGAGAAAAAAGTATAAAATACAAAAAGTCAAATCATGTGGTGAACGATTATGTTCTCAATAGTGCTTTCATTAAAGTGTTTGGGACCATGTATTGTTGGGTAGCATTGCCATACTCCACTGATATGTGGTTGAGAATGGTGATGTG
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 GATGCACATTTAAGGCCGATAGTTTGATGCTGCTTCTTTTTTTCTCCAAGTGGCAGATTTATCACATGTGGATAATAGATATTAGGTTCATCTGTTTGGAATGCATATGTACAGTGAAAGATGTACTATGGCACCGACATCTTATGAAAT
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
278
4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 AATTTAGAGATCACATTGTCTTATGAACATATTGAATATGGTTAAAACTTGTGAATATCAGATTATCAGTTCAGTATAAATTAATTTACTTCTGGAGTTTATCTGTATTGAAATTTATTCCGTAAATTGTATCTGTTAACTGTAGTAGTG
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CTGCAGAAACTCTGCCTGAGGCAGTCCATATCTTTTCTCCTTTTACTACATTGTAGACTACCGTAGACCATATGATCCTGCCATTCTTAACATGCATGCAATTGAAGCAATTTGGCTATGATACTTACTAGATTGCCCAGTAAATATATG
OsCYP40 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CTAATTTTAGGAAAATAGTTGTTATGCATTAACAGTCCCCCGACTAAATTAGTCTAATCTTTTCGTAATGTTTATTTAATTTATTGTGCAGGAATGAATGTTATAACCAGCATTGCAACTATTCCTACCTATAAACCAGCTGAAAGGATC
OsCYP40 -------------------------------------------------------------------------------------------...........................................................
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 CGCTTCTTCAATGATTTTGCCCAGTTGATTGGTGATGAAAGAGCTCAAACTGCTCGAGCGTTGTGGGATCGCCCGCTAAAAACTGTTTATATCAGTGATTGCGGAGAGCTAAAAGTGACCAAACCATCACTTTCCCCTCCAAGTCTGCCA
OsCYP40 ......................................................................................................................................................
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
4660 4670 4680 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g19610 TAAAAAACCACAGAGGCAATTTTGTGTACCTACACTTATTCTTCTCCCTCCAATGTCAACTGTATATATGCCATCTAGTATTAACTGACATCTGCTGTGTTTCTGGCCCAAAATGTGTACGACGGGTCTCTGATTCATCGAAGTAAATAT
OsCYP40 ...---------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP28 ------------------------------------------------------------------------------------------------------------------------------------------------------
4810 4820 4830 4840 4850 4860 4870 4880 4890
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . .
08g19610 ATTACAGTGCTGTGCAAAATTCTTGACCTGTCAGCTTTCATCTTGGATAACCGTTGAGACTATGATGCTAATGATTAGCTTCTCACAGAATTGACCTA
OsCYP40 --------------------------------------------------------------------------------------------------
OsCYP28 --------------------------------------------------------------------------------------------------
Exon II
279
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TTTGAATCCCCCGTGTCACTGTGTCCGTCAGATCGGCCATTGGCGGCTAGAATCGAGGCGGAGAGTAGGGACTTGTTTTTCTGGAAACCCCCTATTATTAGTTTCGTAACACAAATAATTGCTCCTCGTCGCCGGAGCAGAAGGTCTCGA
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GGAGTCGAGACTTCTCTCCCCCAAATCCCCAGAACCCTAGCGGAGCTCCACCTCGAGATGGCGACGGCGAGCGATGCGCCGGCGAGCTCCACGATAACGACCGCCACGGACGACGCCGAGGTGGAGCGCGACCAGGGCAACGGCAATGGC
OsCYP73 ---------------------------------------------------------.............................................................................................
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GCCGTGTCAGCCGCACCCGCCGCCGTGGGGAAGGAGGCGGCGGCTGAGGAGGAGGAGATGATCGGCCCGGCTCCCGTGCCGCCGCGGCCGAGGAAGAAGCGCCCGCTCCAGTTCGAGCAGGCCTTCCTCGACGCCCTGCCCTCCGCGGCC
OsCYP73 ......................................................................................................................................................
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 ATGTGAGTGTTCTCCTCGCCGTTTCAGGTTCGGATTTGGTAGGGGGTGCGTCGGCTGGGAGGCTGACGGCCGTGGCTTGGTTTTGCAGGTACGAGAAGAGCTATATGCATCGGGATGTCGTCACCCACGTCGCCGTCTCTCCTGCTGACT
OsCYP73 ...--------------------------------------------------------------------------------------.............................................................
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 ACTTCATCACCGGGAGCGCAGATGGTAATCTCTCGCGCTCACGTCTCTGCATCGTACCGTATTCAGTGTGCGATACTAGTGGTTTGTGCCTTTGTGGTAGTTTGTTAGCTTGTGGCAGCATGTGTTAGATTTCGGATGGTATCTTGTGGA
OsCYP73 ........................------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 AATGTCAAGCCCTTCTACATAAGGGATAGTCCTTCACTGAACCATAATCACGGGCTTGATAGACTTGCGTAATAGTATTGGTTGAAAAACTTTTAGATCTATGGATTGATCAAATCCTGTGGTATGATTCAATAATAAGCTGATTCATTT
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TCATGTAATGGGAGCTTAATTACCATCCTTTTCTCCTCTCTACCCAGGACATTTGAAATTTTGGAAGAAGAAACCAGCTGGGATCGAATTTGCTAAACATTTTCGATCTCATCTCAGTCCCATTGAAGGCCTAGCTGTGAGTGTTCAGAT
OsCYP73 -----------------------------------------------.................................................................................................------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TCTTATATTGACTTCCCTAATCCAGGGAAAACATTTGCATTCGCAATATATATTAGTCTATTTATCTTGCTAGTTGCTACTCTTAGTCACAGCTCCTGCCTATGGAACTATGAACAACCTCAAATTATAGCGTGATTCAGTAATGCAGGT
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ----------------------------------------------------------------------------------------------------------------------------------------------........
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GAGTGTTGATGGCCTACTTTGCTGCACGATCTCCAGCGATTGGTCTGTCAAGATATATGACGTTGTGAATTATGACATGATGTTCATGATGCGTCTCCCATTCGTTCCGGGTGCAATTGAGTGGGTTTATCGACAAGGAGATGTTAAACC
OsCYP73 ------................................................................................................................................................
OsCYP58 ......................................................................................................................................................
D
Exon I
Exon IIIntron I
Intron II
Exon III Intron III
Exon I
Exon IV
280
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 AAAACTCGCTGTTAGTGATCGTAATACACCTTTTGTTCACATATATGATACTCATTCTGGTTCAAACGATCCCATCATCTCCAAAGAGGTAATCTGTTGTTGCTTTCTGTGTGTCGATGTGTATTTCTTGTGAATTAAAGTAACTAAATG
OsCYP73 ........................................................................................--------------------------------------------------------------
OsCYP58 ........................................................................................--------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 ATTCTTACTGCATGAGCTGCCTCTTTATGCAGATTCATGCTGGCCCAGTAAAGGTTATGAAATATAACCATGTTCACGATGTTGTGATATCTGCTGATGCCAAAGGACTCTTGGAATACTGGTCTCCGTCTACCCTCAAATTTCCAGAAG
OsCYP73 --------------------------------......................................................................................................................
OsCYP58 --------------------------------......................................................................................................................
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 ACGCGTATGTTGTTTCTCTCTTGGAGTTATTTTCAACGTTTGTGTAGCTTGTGGTTTTGTCTTCCCAAAACATTGCACTGAACCACCAAGTACTTGAGCCATGATTACACCTACCATTTCCTTTTCTGTTAAGTCTGCAGCTGGAACAAT
OsCYP73 ....--------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ....--------------------------------------------------------------------------------------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 CCTAGGTGCTAATTAATCAACGAACCAGCTTTTTTTAAAAAAAGTTAGGTACTATTATGTTTGGTGAAGATGCACTTAACTTAAAATCTTTTCTGATTCACTTTTGTTTCTTATAATAACATATTATCCTACTTTAGTATTTATGGATTG
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TTTTTTCATTTCACCTGTCACTCTGGCAGTGTATAAGCTCTGGGTTGGTTCCTTTTGTAATGGCTATATTTATATGTGCTAACAGCTGTTCTGTCCCTCTTGGTGAATGCATCAATCCATCATCTTATCAATGCATATTGCTTTTGTTTT
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TTGTGCAGGGTGAACTTTCGTTTAAAAACAGACACAAATCTATTTGAAATCGCAAAATGCAAGACTAGCGTGTCTGCTATTGAGGTACTTCTTCTATTTGATTCTCAAGTTATTTGCATTTGTTGCTTCATTATTGATAGAGCTACAGGA
OsCYP73 --------............................................................................------------------------------------------------------------------
OsCYP58 --------............................................................................------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TTACTCTGATAAACGAGCTTGTGTTGTCCAGATGAGCAATGACGGCACTCAATTTGTTGTTACATCCCCTGATCGTAGGATACGGGTATTTTGGTTCAAAACTGGGAAACTAAGACGTGTATATGATGAGTCCCTTGAGGTAAGAAATTC
OsCYP73 -------------------------------............................................................................................................-----------
OsCYP58 -------------------------------............................................................................................................-----------
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 CATCTGTACAATGCATGGTACAGATAAATATGTTCTGTTATAATCCCCAACTGAATGCTGTCAATTATAGGTGGCACAAGATCTGCAGAAAAGTGATATCCCGATGTATCGTCTTGATGCTATTGATTTTGGGCGAAGAATGGCTGTTGA
OsCYP73 ----------------------------------------------------------------------................................................................................
OsCYP58 ----------------------------------------------------------------------................................................................................
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GAAGGAAATAGAAAAGACCGAAAATGTTCCACAGCCTAATGCTGTATTTGATGAGAGCAGCAATTTTCTTATTTATGCTACCCTTTTGGGCATAAAGGTATGCTATTTTTTTACTGCACATCTTTTCTTTTATGTAGTATGTGTTTGATA
OsCYP73 .................................................................................................-----------------------------------------------------
OsCYP58 .................................................................................................-----------------------------------------------------
Intron IV
Intron I
Exon V
Exon II
Intron VIntron II
Exon VI
Exon III
Intron VI
Intron III
Exon VII
Exon IV
Intron VII
Intron IV
Exon VIII
Exon V
Intron VIII
Intron V
281
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TCATTATATGTACTTCAGTTATTTATATAATTATTTTTTATGTTCGGAATGATAAATGTTGCTCAAGAAAATATTTGTCCTTGTGGAATTCCATCTTATGGATCAGCTGCTAGTTAAACAGCATGTTTTGTATTAGCTTTCTAGCAGCTT
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GTTTTGTATTCATTATGCTCCTATCTAGCTGTTGAGTTCATGTAAATATCTGGCATGTTGCTATTGTTGAACATATTTCTGTATGATTAATTCTGATTTCCCATAAATATGCAGATTATAAATTTGCACACAAATAAGGTTTCAAGAATC
OsCYP73 ------------------------------------------------------------------------------------------------------------------....................................
OsCYP58 ------------------------------------------------------------------------------------------------------------------....................................
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 CTGGGGAAGGTAGAAAACAATGAACGTTTTCTGAGGATTGCTCTATACCAAGGTGATAAAGGGAATAAGAAGGTTAGAAAAATTCCTTCAGTAGCTGCCAATGTCAATGACAGCAAGGAGCCTTTATCTGACCCAACGCTGCTATGCTGT
OsCYP73 ......................................................................................................................................................
OsCYP58 ......................................................................................................................................................
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GCCTTCAAGAAGCATCGAATATATCTCTTTAGGTACATCAATTCAATGATCTTTGGATAGGTCGTATTGGCTGATTCCGCATGCTGCTTCTGTAGACTCCTTTCTCTCCAGATGGTTTTTCTTAAATTTTAATATCCCGCTTCTCAATCC
OsCYP73 ................................----------------------------------------------------------------------------------------------------------------------
OsCYP58 ................................----------------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TCTTTTTTCATTATACAGTCGTAGAGAACCTGAGGAGCCTGAAGACGCAACCAAGGGTAGGGATGTTTTCAATGAAAAACCCCCACCGGAAGAGCTTTTGGCTGTATCAGATTTAGGGAAGACTGCTACAACATCACTCCCTGATAATTT
OsCYP73 ------------------....................................................................................................................................
OsCYP58 ------------------....................................................................................................................................
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GGTATGTTTTCTCAATCATTTCTAAAAATGGTAATTTGCTACTATTGATTTTATGGAAATTTGATATCTTGGTGGGATAGTCTTTGAACATTTTTCCTTTTTAGGTCGCATTTATTTACATGAGTATTTCCTATTTCAGGTGATGCATAC
OsCYP73 ...------------------------------------------------------------------------------------------------------------------------------------------.........
OsCYP58 ...------------------------------------------------------------------------------------------------------------------------------------------.........
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TTCTATGGGTGATATTCACTTGAGATTATATCCAGAAGAGTGTCCAAAAACTGTGGAGAATTTCACTACCCATTGCCGGAATGGTTATTATGATAATCTCATATTTCATCGTGTGATTAAAGGCTTTATGATTCAGACTGGAGACCCTTT
OsCYP73 ......................................................................................................................................................
OsCYP58 ......................................................................................................................................................
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GGGGGATGGCACTGGTGGGCAATCAATCTGGGGAAGGGAGTTCGAAGATGAATTTCACAAAAGGTATATAAGTTGTATGCCTTTTCCTGTTTTTCTTTTCTCATTTGCAGAAAACAATGCAGTTTTTCATCTTATGTACCGTTTAGATGA
OsCYP73 ...............................................................---------------------------------------------------------------------------------------
OsCYP58 ...............................................................---------------------------------------------------------------------------------------
3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 CAGATGTCTATGCAAGGTCCAGGAAAGATATCTGTATAGTCATGTAAATTTTGCATGCACTGTGCTTATTGTTGTTGAAGAGAACACAATCCATATGAATAATCCTATTTGAATATGCTTATAGTGACAACTGACAGCTATGGCGGCTAT
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon IX
Exon VI
Intron IX
Intron VI
Exon X
Exon VII
Intron X
Intron VII
Exon XI
Exon VIII
Intron XI
Intron VIII
282
4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 CTTGCAGCTTGAGACATGATAGGCCTTTTACTCTTTCAATGGCCAATGCTGGACCAAATACCAATGGGTCCCAATTCTTTATCACAACTGTGGCAACTCCATGGCTAGACAACAAACACACAGTGTTTGGTAGAGTTGTGAAAGGGATGG
OsCYP73 -------...............................................................................................................................................
OsCYP58 -------...............................................................................................................................................
4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 ATGTTGTTCAGGTACCAGCCTTTTTGACCAACTTAGTGACTGTGTGTGAATTTTCATATCCTTTATTGTGCTTCCTAATATGTTTTGTTTTAAACTTAAAAGCAAATCGAGAAGGTCAAGACCGACAAGAATGACAAACCATATCAGGAT
OsCYP73 ...........-------------------------------------------------------------------------------------------................................................
OsCYP58 ...........-------------------------------------------------------------------------------------------................................................
4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GTGAAGATCTTGAACGTTACAGTTCCAAAGACATAAGCTCAAACAATTGGTCAGTTTTTCTGCTCTTGATACCATAATCTAACCTCATAATTCATTTTGTTTTGGGGATTGTCTTGAACAAGTGACTTTGCTTGGTGTTTTCCTACAAAA
OsCYP73 ....................................------------------------------------------------------------------------------------------------------------------
OsCYP58 ....................................------------------------------------------------------------------------------------------------------------------
4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TACATACAATGCAAACTAATCCTTAACTGGCACAGATGTCTTGTGAACATTAATTTTTTTAAATTGTTCCCTTTTCTAAAGTTACCTACTACCTCCATCCACAAATATAAGCATTTCTAGCATTTGAAATTTGTACATTTCTGGAGTACT
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
4660 4670 4680 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 ACTTTCCCAATCCAACCACCGCTGCATTTATTGCTCACTTAGTTGTATTTCTAGGCATGGTATTTTCCCTATCAAGTAGAGTCCCAGGGGCATCAAATTAATTTCACCTAAACCTTATTTTCTGTTAAAATCCTAGAAGTGATTATATTT
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
4810 4820 4830 4840 4850 4860 4870 4880 4890 4900 4910 4920 4930 4940 4950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GTGGATGGAAAGAATATAGTACATACATTAAGATAATACTGCCAGTTGCAACAACATTAAGAATATAGCCTGCTTTACTTATGTGGTTATCAAAGCTAATTTCATAATGGTTTGTTGGGTTAGACCTAAATACTTCTAAATTTACTTTAG
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
4960 4970 4980 4990 5000 5010 5020 5030 5040 5050 5060 5070 5080 5090 5100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GAGGTCATCTTTGCTAGACAACCTGTAGGAAAACCAAGCATCCAGTGACCAGTGGTGCTCTTTATTATTACCTTGTGTTATCCCCTGTTTGCCATGGGATTGTTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTTTTTCATG
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
5110 5120 5130 5140 5150 5160 5170 5180 5190 5200 5210 5220 5230 5240 5250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 TGGACTGTATGAAGAACGGTTCCATTTCTTTATCACCAGTTTCATGTTGACTGTATGGAGAACGGTTCCATTTCTTGTATCACCAGTGCTCTCATGTGGTCATTTCGTGTGCCTTCTGCAGGTTTGACATTGTACGCATGAGGTTCTTCC
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
5260 5270 5280 5290 5300 5310 5320 5330 5340 5350 5360 5370 5380 5390 5400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 ACCCTTTGCAAGCCTGTGAAAGACAAATGTTCTGAACTTTATGTGCTGCTGTGGTGCATCGAGTTGGTCCTTCAAACCTCAACCCAGAGGCAGCACCATATTATTAGAAAGCACTGCCTTTGATGAGGTATGTGTTCCGAGTTTTAACTA
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon XII
Exon IX
Intron XII
Intron IX
Exon XIII
Exon X
283
5410 5420 5430 5440 5450 5460 5470 5480 5490 5500 5510 5520 5530 5540 5550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 GCTTTCCCATCCATAACCCAAGGGCAATGGGACTGTAACATTTTTGCTGTACACGGGATTGGTATTGCAATTTAACAATGGAAAAAATGTAACGAGCCAGTTAGATGAGGATACATGGACGATTTTTCCATGCATGGGTGTAAGGGCGTA
OsCYP73 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP58 ------------------------------------------------------------------------------------------------------------------------------------------------------
5560 5570 5580 5590
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44330 AGTTTTATTTTGACTAGCCACTGAATGCTGATACCAATTAGACTG
OsCYP73 ---------------------------------------------
OsCYP58 ---------------------------------------------
284
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 AAGTGAAGAAGCTTGGGAAGGAAAGATTGATGGATCCAAGGTCAAAAGCCGACAGAATAGTCCGGCGTACGGCGATGATCGGCGCCGCCACCGCCGCCTACTTCCTCCTCACCGCCGATTACGGCCCCGACTACCCCAACCCGGTAGGTA
OsCYP20 -----------------------------..................................................................................................................-------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 ACTAGACTAACTTCATTTTTTTCCCTGTAAAAAAATGTGAAATTGCTGCTCCACTGTAGCACAAGTCCAATACTACCAGTATGTTGGTGCACAGAATTTGTGAATTTTATGAAGCTATGAGCTTTTCTTTCTTGTAGCCGTTCATGTGGC
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GCTGATGTCTGACTGGTCAGGTTCAATACGAGTATCTACTGCATAATACTTCATACTATTGATTATTGAAGTACTCCCTCTAAGTCATTTTAGCATTTTTCATATTCATATTGATGTTAATGAATCTAGACATATAAATCTATCTAGATT
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 CATTAACATCAATATGAATGTGGAAAATGCTAGAATGACTTACATTGTGAAACAGAGGAAGTACTACATACTGAATAAGGAGAGATCTTAATTAGGCCTAGTGTTTTAGAAGTGTTTAAGATGCTGAACTGCTGACAGCTACATCAAAGA
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GCTTGTGTGTGTGTGTGTGTGGGGGGGGGGGGGGGGGCAGATTAGGAAGGCCATGGAAGCATCACCGCAGTATATCAAGCACCTCTCCATCTTCAAATATGGCCCAAGGAAGAACAACAAGAACCAAGAAAATGCAGAGCCGGAAGCCAC
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 CAAAGAGTAGTAGTTGGAATCTGCACTAGTCACTGTATGCATCCTATTGCTGTGTATGTTGTACAGTGGGCACTAAATCTGAATGTCGTCACTGTCTGCATCCCTATTGCTTTGTTAAGTACTGTAGTAGGAGACTCGATCTGTATTAGC
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 ACTATCTAGAAATCTTCGCTTTTGTTTCTTCATTAACCTGCTATGCATGATCCAAGTGGTATCTATGTACTATATCTATCATGAATGAATAAACAGGTAGCCTTTTCACTTTACATTCTTTGAACCAAAAGACAAGGAGAGCAACAAGTT
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GAGTTCACTTGTTTTGTTTCACTTTCACAAGGAAAAGAAAAGAAAACAAAACTTCTCCTCTTCGGTTTCACTTTCACAAGAAAATAAGGAGCAATTCTTATATTTGGTGAGGGGCTTTCTGTAATTTATAGTGTATAAGAATTATGTAGA
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon I Intron I
E
285
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GAAGCCCTCGTATTTGTGGCCTCCCCTCCTCCTCCGAAGAGAGAGAAGGGAATCTCTCCTCTTCTCTTCTCTTCTCGCGCCGCCGCCGTGTCCAGTGCCCAAGATGTGGGGCAGCGCCGACGGCGGCACGCCGGAGGTCACCCTGGAGAC
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 -------------------------------------------------------------------------------------------------------...............................................
OsCYP18-2 -------------------------------------------------------------------------------------------------------...............................................
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 ATCCATGGGCGCCTTCACCATCGAGGTACCATTGGCTCGGCTCGGCTCGGCCGTCTCTCTCTTGAGCTATTCTTTTCGTCGGCATACACCGAGTTAAGTTCTTGCTCCTTGATTTGATTTGGTCGCTTCTCTCGTCGATCGACAGATGTA
OsCYP20 -------------------------------------------------------------------------------------------------------------------------------------------------.....
OsCYP19-3 .........................------------------------------------------------------------------------------------------------------------------------.....
OsCYP18-2 .........................------------------------------------------------------------------------------------------------------------------------.....
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 CTACAAGCACGCGCCCAAGACCTGCAGGAACTTCCTCGAGCTCTCGCGCCGCGGCTACTACGACAACGTCATCTTCCATCGCATCATCAAGGTTTCTGCCCACTCCTTGCTTGCTGCTTTGCTTAATAGGAGTATTAATCAATCCGGTCA
OsCYP20 ...........................................................................................-----------------------------------------------------------
OsCYP19-3 ...........................................................................................-----------------------------------------------------------
OsCYP18-2 ...........................................................................................-----------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GGTTTTCTTTACTGCTTTTATCTTCTTGCGGTTTAGGATTTCATCGTGCAAGGTGGGGATCCTACTGGCACCGGAAGAGGCGGCGAATCCATCTACGGGTCAGCATATTTCTTGCTTTCCTCCTCTTTCAATTTGTAAGATTGCATTGCA
OsCYP20 ------------------------------------..............................................................----------------------------------------------------
OsCYP19-3 ------------------------------------..................................................................................................----------------
OsCYP18-2 ------------------------------------..............................................................----------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 ACTGTAACACTCGATTTTGAACTGTATTTTGAGCTCACATCATCAACCCATGATGGATAGCACTGTATTTCTACCTGTTAAAAACATAATAGGGAATTAGTACCTATTGTGTAGCAGCATTTGGTAGCGATTGTACCATTGTTGCTGGGC
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 TCCTGTAGCATCTTGTTTGTAAAAGTAGCTAAATTTATTATTCCTTGTTATTTTCCACGCCACGCCGTTATTGCATTCTATTTGGGGCTCTGTTTGAATCCTTACAAATGTTTGTGTTCTTGCTGTGTTTTTACAGAGCAAAGTTCGAGG
OsCYP20 ----------------------------------------------------------------------------------------------------------------------------------------..............
OsCYP19-3 ----------------------------------------------------------------------------------------------------------------------------------------..............
OsCYP18-2 ----------------------------------------------------------------------------------------------------------------------------------------..............
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 ATGAGATTAGGCCAGAACTGAAGCACACCGGCGCTGGAATTCTGTCGATGGCCAACGCTGGTCCAAATACAAATGGAAGCCAGTTCTTTATTACTCTTGCGCCTTGTCAGTCACTTGATGGTGAGTCTACCGTACTGTCTCATTGTTGCT
OsCYP20 ........................................................................................................................------------------------------
OsCYP19-3 ........................................................................................................................------------------------------
OsCYP18-2 ........................................................................................................................------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 CCAGTTTTACATTATGCTTTCGCTTGTTTGCTGTCTATGAATGTTCTTTGCATTGCCCTGGAAAGCTGAAGTTCACTCTGTAGGAGTTAATCAACAATTTCTCAGATTGCAACCTAAAAACTTTACTATCAAATCACTGCAAGTTTTTTT
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon I
Exon I
Intron I
Intron I
Exon II
Exon II
Exon II
Intron II
Intron II
Intron II
Exon IIIExon III
Exon III
Intron III
Intron III
Intron III
Exon IV
Exon IV
Exon IV
Intron IVIntron IV
Intron IV
286
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 TTAATATTAGATTTGGAATAATTACAAGAAAAACTTGGGACTAGCTTTTGAAGAGCCTGTTGTAGAGTGACTTTGAGTCTAGTGCAATCACTAGGAGCGTGATTCTTGGAGACTTCTGGTGGATTTCTGGGACTCCTGTTCTTCTGTATT
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GTGATGTTGGTACATTGGACTGCATTGTTGTTAATGTGTAATGCAATTGTGAAGGACTTCTGCTAGCACCATCACTGTTTTTTCTTCCTAATTGCCAACCCTGGAAGATTTTGCCCCTCAGGATGTCACTGCTTACATCTGTTTTTGACA
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 TTGTATTTCGCACTCTGGATTGCTTTTACAAATATGTTCTTTTCGAAATTTCCACATTGAAAGTCAGGTACATTTTCATGCTATTAAGGTTATATGGTTTTCCTATTCCAGGAAAGCATACAATTTTTGGGAGAGTATCAAAAGGAATGG
OsCYP20 ---------------------------------------------------------------------------------------------------------------.......................................
OsCYP19-3 ---------------------------------------------------------------------------------------------------------------.......................................
OsCYP18-2 ---------------------------------------------------------------------------------------------------------------.......................................
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 AAATTGTCAAGCGCCTTGGAAGTGTCCAGACAGACAAAAGTGATAGGTAACACCAAAACTATATGCCATGGTTGTACATATCACATAATAGATATAGCATCCGATTTGTCCTGAGGGAGAGTTGAGTGAGTGCAACGCCTCGCTATTACC
OsCYP20 ..............................................--------------------------------------------------------------------------------------------------------
OsCYP19-3 ..............................................--------------------------------------------------------------------------------------------------------
OsCYP18-2 ..............................................--------------------------------------------------------------------------------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 ATGCATATGCAATGGCAAAGTAGGTAGCAGGGTAGCAGCAGGAGGGAAATATTTGGGATGATGAGTTGCAGCGTTAGTAGATTTGGCTAGGATCTACTCCCTTCGTTCCACATTATAAGATGTTTTGGCCTCTCCATTCGTTGGCTAGAT
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 TCACTAACATCCATAAGCAGTTTAGCACATACATGGATCCATGCATTAATCTATCCAAAACTCAAATCGTCTTATAATTTTAACTTATAATGTGAAACGGAGAAAGTATTCATCATGGTAGAGAACAATGAGAGCTGCGGGTGAGTGAGG
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GCGAATGGTGGGAGGGAGATAACCAAATCATGTGTACAATCCATGTATGGTGCCTGTAAAACTGAAAGGGAAAATGATCGATATGCATTTTTCCTGTCGAATTTATTCAGACTTGTTATCTTTGTTGATTCAGGCCCATCCATGAAGTGA
OsCYP20 -------------------------------------------------------------------------------------------------------------------------------------.................
OsCYP19-3 -------------------------------------------------------------------------------------------------------------------------------------.................
OsCYP18-2 -------------------------------------------------------------------------------------------------------------------------------------.................
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 AAATCCTGCGGACTGTGGTCAAAGATTGAGGCACAGCGGGTCTACTCGGTGTCACTCATGACTTCTGGTATTTGGAGTTGTAGCTGCTGTCAGTTTCAGGAATTGTTTCCTGCTTGTTCCGTGCTTATGCAGTGGTGTATCGTTTCGGAT
OsCYP20 .............................-------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 .............................-------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 .............................-------------------------------------------------------------------------------------------------------------------------
Exon V
Exon V
Exon V
Intron VIntron V
Intron V
Exon VIExon VI
Exon VI
287
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
08g44520 GGGCTAGTTTAGTTATTAACAACTCATGAATCGTGATCCGTCTTAATTATTCTCACCTGAACGAGTAGTGTTCAGTGATTATCAGTGGAAATAACTTTCGAAATATATGAAGTCATTTTGAGCGCATGATGTAATTCAGCTGATATTCAG
OsCYP20 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP19-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsCYP18-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
3760 3770 3780 3790 3800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .
08g44520 CATGTTACATGCTGTACCAACTCAGGGATTTTTTTTTTATGTTCAAAACATAAA
OsCYP20 ------------------------------------------------------
OsCYP19-3 ------------------------------------------------------
OsCYP18-2 ------------------------------------------------------
288
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 ATTCCTAAAACGGAGCCGAGGACGCGAGGCGAGAGCCGTATATCCGCTTACGTCACCTCGCTGCTTGGCCTTGTTTCGCCAAGCCGCCCCCAGACCCAGACAAGTCGCAGCCATGGCGATCCCCCGCATCTCGCCGCGCAAAACCCTGCC
OsPDIL2-2 ----------------------------------------------------------------------------------------------------------------......................................
OsPDIL2-2a ----------------------------------------------------------------------------------------------------------------......................................
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 CCTCTTCGCCGCCCTGGCCCTGGCCTTGGCCTGGGCCTTCGCCGCCCCGGCGTTCGCCGACGGCGACGACGTCGTCGCCCTCACGGAGTCCACCTTCGAGAAGGAGGTCGGCCAGGACCGCGGCGCCCTCGTCGAGTTCTACGCGCCCTG
OsPDIL2-2 ......................................................................................................................................................
OsPDIL2-2a ......................................................................................................................................................
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 GTTCGTTCTCGTCCCCTTTGTCTCGCCAGATCCCTGCGTAATTCGGCTGGATCGGGTCGGGTCGGATCGGATCCGTGTGTGGGTCGTGAATAGTGATGCGGGATGCGAGGTCTGGGGATCTCTACGTTTTTGCGGGGTTTAGCGGACTTT
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 GACTCTTAGATTTTTTCTTCTCTCTCTCTATATATATTTCTCATAATTTGCACTTATCTTATGAAATTGATTGGATAGTTATAAACTTGCTGCCTTACTGGAAAGAATCATCCATACGAATTCGATCCTTTTTGTCTATCTGGATTTAGA
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TTGTCAGCTGTAAATTTGTTTCGACTTGTAGTATTTTAAATCAGGTATTGATGGGTGGAAATTTAATACTGTTGTTGGTTACAAAAATTAGAGTATTTCTATAAAAGCAGGGCGCATTCTAAGCCTTTTATCTAAAAAAACAAAAATTAG
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 AGTACACATGGCAAATTTTTTGTTAGGGTTTAAGCAGGAGGTTTAAATGTGCCTTGTAGCTTAGTTACTTGTTGTGCAGTCTACTTGTGCTCTACTTCTACTAGTGACGTAAATCCAATTTGGCATTTCATTTTTTAATTAATACCTGAC
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TCTGTTTATGATATCGCAGTTTAGGTTTTGTAAAATTCCGGCTAATTTTGCACTTGCCTCTTGATGGAAATAATAGAATACCATAACTTTGTAATTATGCTACTATCTATTAAATGGATTCACAAAAGAAGCAGGCCAAAATTATATTTT
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TCCTATTTCATGACTGCTAAGGGGTGCGATTCGGGATATTTATACTGTGTTAGAAACAAAACACTGCTGTAAATACCTAGTAACTTGTATCGATTGATTTTTGCTGAATCCTGTAAATGCAGCAAGTTTTAGTTCATTATATCTTGAGGG
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 AGTCTTGTGAGATGTTGAATGCGATTATATGAATATAACTGATCTGGAGATATTCATAGAGAAGCTTCTATAGTTTGCTTATCACAGTGCTCGCGTGGCCATATTTGCTCCTGTTGTTGTGCCTTCTCAGATGTATTACTAGAAAAACCA
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon I
Exon I
Intron I
Intron I
F
289
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TGCTTAAATATTTGTGCATCAGTTCAGCATTTCAAAGATATCTAGTGTTACTCATCGGTCTATCTATCTGGTTAGGTGTGGTCACTGCAAGAAGCTTGCCCCTGAGTATGAAAAGCTTGGTGCAAGTTTCAAGAAAGCCAAATCTGTCTT
OsPDIL2-2 ---------------------------------------------------------------------------...........................................................................
OsPDIL2-2a ---------------------------------------------------------------------------...........................................................................
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TATTGCAAAGGTATGTTCTCAAGTTCAATACCTGCTAATTATTTGGGCATTGCTTGAAAATATTTTCTCATCTCCATCCTGCTACTCAGAGGTGGTCAGAAAATGGTTAGAACATGCATATTCATGAAATGTGTTGCCATCCTCATTTTG
OsPDIL2-2 ..........--------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ..........--------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 ATTTGGTTTGATAAAATTAAGGATTCTCACTTCAACTCTATTTCTCGCTTTAGGTTGATTGCGATGAGCACAAGAGTGTGTGCAGCAAGTATGGAGTTTCTGGGTACCCAACAATTCAATGGTTCCCCAAAGGATCCCTGGAGCCCAAAA
OsPDIL2-2 -----------------------------------------------------.................................................................................................
OsPDIL2-2a -----------------------------------------------------.................................................................................................
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 AGTGAGGCTGCATAACTTTTGTTCAAATACTTTTGAACATCTACACCAGACAAAATTTTCATGATTTATATCACTACAACCATGTAGGTATGAAGGACAACGCAGTGCAGAGGCCCTTGCTGAATTTGTTAACACTGAAGGAGGTAACAC
OsPDIL2-2 ...--------------------------------------------------------------------------------------......................................................-------
OsPDIL2-2a ...--------------------------------------------------------------------------------------......................................................-------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TTCAAACTTATAATAATAGAGTTAGACGGTTAGTAGCTGGTATGGTGCCATGGGCTGGGAGTTCTGGATATCTCATATTTCCTTGAAAATTCTGTTTGGTGTGCAAGTCTTAGTTTCCACAAACAGCTTCTTGAGTTAATTCATTTTTGT
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 ATTTATCTCTTCACTCGGTTACAACAGCATATAATCATGAATTTTCCTGATTTCAGGCACTAATGTAAAGCTGGCAACCATTCCTTCAAGTGTTGTAGTGCTTGGCCCAGACAACTTTGACTCAATTGTTCTTGATGAAAACAAAGACAT
OsPDIL2-2 --------------------------------------------------------..............................................................................................
OsPDIL2-2a --------------------------------------------------------..............................................................................................
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TCTTGTGGAGTTCTATGCGCCATGGTATGCTTTTGTAAAGTTGTCTTACTTTTTTCCTCCATAATCTTGACACAATTTTTGCATGATTTTTCATGAATGAATTTTGACTGGATGCTAATTTTGATCCATAGGTGTGGCCACTGCAAGCAT
OsPDIL2-2 ........................-----------------------------------------------------------------------------------------------------------...................
OsPDIL2-2a ........................-----------------------------------------------------------------------------------------------------------...................
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 CTTGCTCCGGTTAGTGGATAACTCTACCCAAAGTTTGTTCTACATGTTATGTTTACTTTTGAAAGTTTCAGTTGTTCTCGACGTGCCAACTAATATTCTTGTTATTATTTTAGATATATGAGAAGCTGGCTTCTGTTTATAAGTTGGACG
OsPDIL2-2 .........--------------------------------------------------------------------------------------------------------.....................................
OsPDIL2-2a .........--------------------------------------------------------------------------------------------------------.....................................
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 ATGGAGTTGTAATTGCTAACCTTGATGCTGACAAACACAAAGACTTGGCCGAAAAGTATGTTCTGACTTATCCGTAATGTTATCATCTGCGTTCTGTTTTTCCTATTGCTGTTAATCTTGTGCCCTGAGATGCTACAGTTTTCCCATGGA
OsPDIL2-2 .......................................................-----------------------------------------------------------------------------------------------
OsPDIL2-2a .......................................................-----------------------------------------------------------------------------------------------
Exon II
Exon II
Intron II
Intron II
Exon III
Exon III
Intron III
Intron III
Exon IV
Exon IV
Intron IV
Intron IV
Exon V
Exon V
Intron V
Intron V
Exon VI
Exon VI
Intron VI
Intron VI
290
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 CACAAAACAACTCATGTTATGGGCATGCTTCCAGCACTTTAAACTCATGTCTTGTTCTATATTTTGATTTCATGATGCACTACATAAGTTTTGACTTGCTTTATGACTTTTAGATATGGAGTTTCTGGCTATCCTACATTGAAGTTCTTC
OsPDIL2-2 -----------------------------------------------------------------------------------------------------------------.....................................
OsPDIL2-2a -----------------------------------------------------------------------------------------------------------------.....................................
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 CCAAAGGGAAACAAAGCTGGTGAAGATTACGATGGCGGCAGGGAGTTGGATGACTTTGTCAAGTTCATTAATGAGAAGTGTGGTACCAGCCGTGATACTAAGGGCCAACTTACTTCAGAGGTTGGTTGAATGCATATAAAACTTCTTTGC
OsPDIL2-2 ........................................................................................................................------------------------------
OsPDIL2-2a ........................................................................................................................------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 ATGGGTGAGCTCATGCAAAAGACTACCTGTTCTGACTATACATTGTTTATTGTTGTGGTAGGCGGGGCGCATAGCAAGTTTGGATGCCCTCGCAAAGGAGTTCCTTGGTGCTGCCAATGACAAGCGGAAGGAAATCCTCTCCAATATGGA
OsPDIL2-2 -------------------------------------------------------------.........................................................................................
OsPDIL2-2a -------------------------------------------------------------.........................................................................................
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 AGAGGAGGTTGTGAAGCTCAGTGGTTCTGCTGCAAAGTAAACTCTAGTCTAAGTTGCATTTATTATTTTTGCAATGACGTTATCTGTACATGCAAGTTGAACCTTGGCATTTACATATTTAAATCAGGCATGGAAAGGTCTACATTGCCA
OsPDIL2-2 ....................................-------------------------------------------------------------------------------------------.......................
OsPDIL2-2a ........................................--------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 TTGCAAAGAAAATCCTGGACAAAGGTCACGACTATACCAAGAAGGAAACAGAGAGGCTTGAACGCATGTTGGAGAAGGTGGGAAACCGTGATCGCCAATCCAAACTTTACTATCACTTGTCATCTTACATAATGAACGACTTGTACTTAT
OsPDIL2-2 .............................................................................-------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 ACAATACTTTTTTATTTTTTTTGCTTCCTGCAGTCCATCAGCCCATCAAAGGCCGATGAATTCATCATCAAGAAGAACGTCCTTTCAACCTTCTCTTCCTAAGGCGATGTCAAACTCCATTCTTATCTTTGGGATATAGGTATAAGGGAA
OsPDIL2-2 ---------------------------------.....................................................................------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os01g23740 AATGACAGAAAGATTAGGAATGTATTCATGAGAGGCCGTTGGCGGTAGAACTAATTTTGTGGGTTTTCCCTTAAGCATACATGTATCCTATTGCCTGGTGCGGATGTTTTAATTCTTTGCTCATGGTTACATGGATTCAGTTTGTAGTAA
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------------------------
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .
Os01g23740 AAAAATAGGGGTTTACTGTCAGGTGGAGAGAAAGATGTTAACCTTGATACATGTTACGACATTGAATTTCAGTCTGATAATAAAGTACCCTCATGGGTCTGAATCGCTCAAAGCTGAATGCACCAAATGGTG
OsPDIL2-2 ------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-2a ------------------------------------------------------------------------------------------------------------------------------------
Exon VII
Exon VII
Intron VII
Intron VII
Exon VIII
Exon VIII
Intron VIII Exon IV
Intron IV
Exon X
291
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 AGTTTTAAAATGGAAAGCTTGCATCAGGCCACGTGGCGTTGACAAAGAACCAATCCCAATGTGGCCACGAGCCCCGGCCACGCCGCCGCCGCCGCCATGGCCATCAAAACCTAGTGCCGCCTCGCGCAGCGCGCTGCGTCGTTTGGATCT
OsPDIL1-3 ----------------------------------------------------------............................................................................................
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 CGACGACGGCCGGCGCCAAGGCACGGAGGGAGAAGAAAACCACGCGCCTCTTCTGTGTTCTCCGGCGATGGCGAGCTCCACGGCATTTGCAGCGGCATTTGCGCTGCTGCTGCTAGCGTCGTCGGCGGCGGCGGAGGGGGAGGCGGTGCT
OsPDIL1-3 ......................................................................................................................................................
OsPDIL1-3a -------------------------------------------------------------------...................................................................................
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GACGCTGGACGCCGGCAACTTCACGGAGGTGGTGGGGGCGCACGACTTCATCGTCGTCGAGTTCTACGCCCCATGGTACGTACATTGTACAGGCAAACCCCATTCCTCGCCGCCGGCTCGGCGCCATGGCGCCTTGCTGATTTTGACCTT
OsPDIL1-3 .............................................................................-------------------------------------------------------------------------
OsPDIL1-3a .............................................................................-------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GAGCTCAGCTGACCAAATCCTCTTCGCCGCCGCCGCAGGTGTGGCCACTGCAACCAGCTTGCTCCGGAGTACGAGGCGGCCGCCGCCGCCCTGCGCTCGCACGACCCGCCGGTCGTCCTCGCCAAGGTCGACGCCAGCGCCGACCTGAAC
OsPDIL1-3 ----------------------------------------..............................................................................................................
OsPDIL1-3a ----------------------------------------..............................................................................................................
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 CGGGGCCTCGCCGGCGAGCACGGCGTGCAGGGCTACCCCACCATCAGGATCCTCCGGGACCGCGGCGCCAGGTCGCACAACTACGCCGGGCCCCGGGACGCCGCCGGCATCGTCGCCTACCTCAAGAGGCAGGCCGGCCCGGCCTCCGTC
OsPDIL1-3 ......................................................................................................................................................
OsPDIL1-3a ......................................................................................................................................................
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GAGATCGCTGCCTCCGCCTCGCCGCCAGCGGCCGACTCCATCGCCAACGACGGCGTGGTCGTCGTGAGTTTGACCTCCTCTTCTCTCTGCATCAGCTGTTTATGCTTATACTTACAAGCTAAAGGCTCTCATTGGATGGCCTAAATAGTC
OsPDIL1-3 .................................................................-------------------------------------------------------------------------------------
OsPDIL1-3a .................................................................-------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 AAAGAATAAGTCAAATTTTAAATTTTCAAACTTAAATTTAAGATTGATTTTAAGATATTTTTAACGTAATTTCTTTTTTAATATTGGCTTTTAAGTCATCAAGAACATATATATATATATGAAAGTTTTATCTATAAATTTATTTTTATC
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 CCCTAATAAGCTGTTTTGGCTTATTAGGGAAAGAGCCAAACGATGGGAGACTAAAATTTAATTTTACATTTGAATTTTTGGTTTTTTATTATAAATAGCATTTACTTTTTTAAGTACATTTGATTTTTTGTTTTTTAATTATAAATAGCA
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TTTACTTTTTTATATGTATATATAAAAGTTTTACTCACAAATAATATTTTTATTTACAAATAAAGTGATTCGGATAAACATACAAATAAGCGAAACAATGACCCGATGCATCAGTCAAATACTCTTGCTGATGCAGGTCGGAGTTTTCCC
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------............
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------............
GExon I
Exon I
Intron I
Intron I
Exon II
Exon II
Intron II
Intron II
Exon III
Exon III
292
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 CGAACTTAGCGGCAGCGAGTTCGAGAGTTTCATGGCGGTGGCGGAGAAGATGAGGGCCGACTACGATTTCCGGCACACGACGGACGCCGGCGTCCTCCCACGGGGTGATCGGACGGTGAGGGGGCCTCTCGTCCGCCTCTTCAAGCCCTT
OsPDIL1-3 ......................................................................................................................................................
OsPDIL1-3a ......................................................................................................................................................
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 CGACGAGCTCTTCGTTGACTCGCAGGTACACTAGCTGAAAATTAATGCTACAGAGATACTCTGCTCTGTTTTGATCCCTCCAGGGGAGGGGTATCTCTGTATTCTTGTTTGCTTAAAATCATCCAAGTAGTTATGATTCATGAAAATTTC
OsPDIL1-3 .........................-----------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a .........................-----------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TGTAAAAAGTTGACAGCGTTCGTACAAATACATATACCACCTCTAAAAAAATGTAAGATCCAAACTCAACTTACACATTGATATATAAAAAACATAAATTCGGTATATGAATAATAGGGTAGTGGTAGTGTCCATACCCGAATTTATCTT
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TTTTTTTCAAGGTGCATGTCAAATTTAAACTTAATTTCTGGTGAATCGATAAATATCATTATACTTTATATTGCCAATTTTTTCAAAAAAATTTACAGCTATTTACAATAAATTTGGAACAAAAATATACACGAGGGGATATGAATATCC
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TCCAAGGGATTAGCTAGTAGTAGCTCACATTTTGGATAAGAAATGGACAGAAATTTGGATTGCTTCTTGTTTTTATTTTTCAGGATTTTGATAGAGATGCGTTGGAGAAGTTTATCGAGTCTTCTGGTTTCCCTACAGTGGTTACCTTTG
OsPDIL1-3 -----------------------------------------------------------------------------------...................................................................
OsPDIL1-3a -----------------------------------------------------------------------------------...................................................................
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 ACACTAGTCCGGCAAACCAGAAATACCTTCTGAAATACTTCGACAATGCCGGCACCAAAGTAAGGACAGACAATTGGTTGACTTCTCACTCTGTTTGCCTACACTGGACTGTTTAGCTTGTTGTTTAGGCCATGATTGCCTCTTATCATG
OsPDIL1-3 ............................................................------------------------------------------------------------------------------------------
OsPDIL1-3a ............................................................------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TGCACACGATTTTCACTGTCTCTCATGTTTTATTTCAATGTGCTGCTGGAATCCATAATTGAGAATGCAACTCTTGTCTCACTTGGTCTGATCATCTGTTCTTTCTGAACCTGATTTTGAGCGTGCAGGCGATGCTTTTCCTGAGCTTCA
OsPDIL1-3 ---------------------------------------------------------------------------------------------------------------------------------.....................
OsPDIL1-3a ---------------------------------------------------------------------------------------------------------------------------------.....................
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GTGATGACAGAGCAGAGGAATTCAGAACTCAGTTCCATGAAGCTGCAAATCAATACAGCGCAAACAACATAAGCTTTCTGATTGGTGATGTCACTGCCTCTCAGGGTGCTTTCCAGGTAATCTTTTGCCTTCTTTTTTCCATAACAATCT
OsPDIL1-3 ....................................................................................................................----------------------------------
OsPDIL1-3a ....................................................................................................................----------------------------------
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TTATTAGTACATTGCTTTGCCCCAACATTTCGTATAAAAACAATGTCCAAGAGTTGAAAATGCCTGCTGTCTGACCATTTTTGAATGCACAATGTTCCAGTATTTTGGGCTTAAAGAGAGTGAAGTGCCCCTCGTTTTCATACTAGCATC
OsPDIL1-3 ----------------------------------------------------------------------------------------------------..................................................
OsPDIL1-3a ----------------------------------------------------------------------------------------------------..................................................
Intron III
Intron III
Exon IV
Exon IV
Intron IV
Intron IV
Exon V
Exon V
Intron V
Intron V
Exon VI
Exon VI
293
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GAAGTCGAAGTATATCAAACCAACTGTGGAGCCTGATCAGATCTTGCCCTACCTGAAGGAATTTACAGTAAGCCAATTTCTGAACTGACGCTATTATCCTCTTCCTCGTTTACTAATTCCCTGATATATATTCTGTGGGTTCTTTAAGTC
OsPDIL1-3 ...................................................................-----------------------------------------------------------------------------------
OsPDIL1-3a ...................................................................-----------------------------------------------------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GCTTGCGTGATATTTAGTCTCACTATTTTATACTAGGGACTATGAAGGTTACTTATTATTAGTACTACCTCTTTTTTTAATTTCTGATGCTGTTAAATTTTTGGGATAAGTTTGACCATCGTCTTATTCAAAAAATTTATGTAATTATTA
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TTTATTTTTATTATGACTTGATTTATCATCAAATGTTCTTTATGCATGATTTAAATGTTTTCATATTTGCATAAAAAAATTTAAATAAAACGAATGGTCAAACATTGATAAAAGGTTAATTACGTCATACATTAAAAACCGGAGAAAGTT
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TCGCTACGTGCTTAGTCAGTTTGCCTTGATTTTCTTTATGCAGGAAGGAACATTGGCACCCCATGTTAAATCAGAACCCATTCCGGAGGTTAACGATCAGCCTGTTAAGACCGTTGTTGCTGACAATCTTCGAGAGGTGGTTTTCAACTC
OsPDIL1-3 -------------------------------------------...........................................................................................................
OsPDIL1-3a -------------------------------------------...........................................................................................................
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 CGGCAAAAACGGTATGTTCTTGGCTACTTGCTTGAAGAACTTGTATTAATTACCACTCGACCTGTTACAGAATGCAAGATGTTTTGGTTTTTGTTACAAGCAATTTTTTTTCAAGTTTAACCGAGTACATAGAAAATGTAGCAACATTTA
OsPDIL1-3 ...........-------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ...........-------------------------------------------------------------------------------------------------------------------------------------------
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TAACACCACATTAATTTCATTAAATCTAGCGTTGAATATATTTTTGTAATATATTTTTTTAGTGCTGAAAATGATGTTACGTTTTTCTTAAACCTGGTCAAACTTTGACTTGGATAAAATCAAAACACCTTATATTAAAGTGGAGGAGGT
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 AGATATTTGTTTAAGTTATGCCCTGCATGCCTGATGCCTGCTTCGTCGTCAACATCTTAACCATTTTGTTAATTTGTTTGCTCAAGCTGTCAAAACATAAGTTCGGTCTCTGTTTTTTTTTTTTCAAATTCCTTTTGCAGTTCTGCTCGA
OsPDIL1-3 --------------------------------------------------------------------------------------------------------------------------------------------..........
OsPDIL1-3a --------------------------------------------------------------------------------------------------------------------------------------------..........
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GTTCTATGCTCCATGGTGTGGACATTGTCAGAAGCTGGCCCCGATCTTGGAAGAGGTTGCAGTTTCATTGAAAGATGATGAAGACGTCGTCATAGCAAAGATGGTAATCTACCCTACCTATGAGCTCTCTCCATTGCTTGGTCCATGCTG
OsPDIL1-3 .......................................................................................................-----------------------------------------------
OsPDIL1-3a .......................................................................................................-----------------------------------------------
3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 GCCTTGTCAAAGATATGATTGAAAAATGCCTTCATAGCATGCATCCATAAACCATCTGATCTATTTATTTCTTAAAAAAACAAAACAAAACAATCAGACTGATTCATTTTATTTTTTTTAGAAAAAAAGCAACCATCAAATTCACATGTC
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
Intron VI
Intron VI
Exon VII
Exon VII
Intron VII
Intron VII
Exon VIII
Exon VIII
Intron VIII
Intron VIII
294
4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 ATGCCTGGGGAAAAAAAAGACTCAAGTTTGACATTTTTTTTGCCTCAAATTCAACAAACCAGATTATTATCAGAACACGCGCGTTTCTCACCGTTCACCTCTTGACCAATTCGTTTGCCCTGAATTGCAGGATGGTACTGCCAACGATGT
OsPDIL1-3 ----------------------------------------------------------------------------------------------------------------------------------....................
OsPDIL1-3a ----------------------------------------------------------------------------------------------------------------------------------....................
4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 ACCGTCAGATTTCGCGGTGGAGGGGTACCCAAGCATGTACTTCTACTCGTCTGGGGGGAACCTCCTGCCCTACGACGGGAGGACAGCCGAGGAGATCATCGATTTCATCACCAAGAACAAGGGCTCCAGGCCTGGGGAAGCAACCACGAC
OsPDIL1-3 ......................................................................................................................................................
OsPDIL1-3a ......................................................................................................................................................
4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TGAATCTGTCAAGGATGAACTGTGATGGCCACTTGGTCAGTGATTCAGCGACTCCAAGTTTTTTTTTTTTGTTTCTTTACCAAAAGAGAGCTGCATTTTGCAAAACACTGATTTATTCAGGATGAATTGCAATCTAAAATCATCTTTGTA
OsPDIL1-3 .........................-----------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a .........................-----------------------------------------------------------------------------------------------------------------------------
4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g34940 TTATTTTTTGTAGGGGGAGTGGGGAGCTTCAGGTTAGGAGGTGCAGTGCAGCTGACAGCTTGAGGACATAGCTCAGTACTGAATTTAATCTTGTATTCGAGTTCCAGGTTTCCAAGTACATGTAGCATTACCAATGGTTTCATACATACA
OsPDIL1-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL1-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
4660 4670 4680 4690 4700 4710 4720 4730 4740 4750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .
Os02g34940 TGTCCCCCCCCTTTTTTTTTTTTGCACCGCTGAAATCTGTTATTGTAACATCATTACACTACATCGAATAATCCCCATTTTAGCTTGCCAAACGAGTAACT
OsPDIL1-3 -----------------------------------------------------------------------------------------------------
OsPDIL1-3a -----------------------------------------------------------------------------------------------------
Exon IX
Exon IX
295
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 CCCAACGCCGGCCCGCCCATCGCAGCCCAAATCCCATGGCCGCCGCCGCCGCCGCCGCCGCCGCCGTGACCTCCCGGCGCTGAAGGCATCCAGCGGCCGCGCTCCAGGCCCCCCCCCCCAGATCTAGGATTGAGCGATCCCCTCCTTTTC
OsAPRL3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 CCCGCGGTCCCCCATGGCGACGCGGCTGCTGTGTTGGACGGCGCTCCTCCTCCCCATCATCGCCGCCACCGCCGCCGCCTCGCCGCTTCCCGAGGCGTGCCCGGTGCCTACTGCGGCGGAGGAAATCCTTGGGCCTGGCGGCACATGCAC
OsAPRL3 -------------.........................................................................................................................................
OsAPRL3a -------------.........................................................................................................................................
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 GACGCTGGATCGCCGCGGCGACCCTGTCGGCGTCATCGAGGTGAGCTGTTGCGATGGCGTGATATAAGTAACAACAGAATTTATTTGTTATATGCATACTGGTTTCCACCATGTTTAGCGATGCTATATGATCGTATTGTGCGAATCAAG
OsAPRL3 ........................................--------------------------------------------------------------------------------------------------------------
OsAPRL3a ........................................--------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 GTAGACTGCCTATTTGCCTAAATGATTGATTGGTTAGTGTTTAAAGTGCGAATATTTGGGGTCCTGAGATTAGGAAGCTTTCTAGGGTTCATACCTATTCCAGTGGTGGTTCTGAGCCCCTGAGATTACGGGCGTAACTGAATCGATAGA
OsAPRL3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 TTGTTACAGTGTTTAGCACCTTGCTACGGGCGTAACTGAATCGATAGATTGTTACAGTGTTTAGCACCTTGCTCTGACGTCTATTCATTAATATATTGCTTATGCCAAAACAAGCGAAGGCCAATTTTATACCAGATTTTGTTTGCTTGC
OsAPRL3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 TTTGGAGATATAATCATGATTCGTTCCATGTTTGTACCACACACATCCCTTCTGAAGCAGCCAAACTACCAAAGGTCTACTATATGTTGGGGTTCCAATCTGCTTTCATAAGCCTTTTTTCCGGCTATGAACCTTGGAAGCCATTTGCTT
OsAPRL3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 GTTTGATTAGTAGAAAATACAATACGAGTTCCTCTGCGGGTGGTATTTTGTTTGTCTTGTCATGTTTGGAGCTATGGTTCCCACATAAAATATCTGTAGCTTGACCCGCTTCTTGTTCTTTTGCATCAAAGCAAGCTCAAATTAGTTTTA
OsAPRL3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 ACTGTTAGATAGTGATTATGGTATCTATTTCTCTCCGTCTTATATCTATTGTAGCTCTAAATGCCAAGCTGACTTTTAATCCTTGTCATCGCAGGGGGATGAGGTAACATTGGCGAAGGCCATTACTCTTCTTCACATGAACAAAGATGA
OsAPRL3 ----------------------------------------------------------------------------------------------........................................................
OsAPRL3a ----------------------------------------------------------------------------------------------........................................................
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 TTACATTGCTGTACTCTTCTATGCTTCCTGGTGCCCATTCTCACAAGAATGCAAGCCAAATTTTGAGATATTAGCATCTTTGTTCCCATCTATTCGGCATTTTGCGTTTGAAGAATCCTCAATCAGGCCAAGGTATGTTATGTGTGCCTC
OsAPRL3 ....................................................................................................................................------------------
OsAPRL3a ....................................................................................................................................------------------
H
Exon I
Exon I
Intron I
Intron I
Exon II
Exon II
Intron II
Intron II
296
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 TGTGTAAATAAAAATGTGCCTCTTAGTGTAAGATGTTCTTTTGGTGAAATGTTAACTTGATTTTTGGAACTGCAGTATAATATCAAGATATGGGATTCATGGTTTTCCAACACTATTTCTCTTGAATTCAACTATGCGAGTGCGGTATCA
OsAPRL3 ---------------------------------------------------------------------------...........................................................................
OsAPRL3a ---------------------------------------------------------------------------...........................................................................
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 TGGACCACGGACTGTTAAGTCCCTGGCTGCTTTCTACCGTGATGTTTCAGGTGCACCAATTATTACATGTGGAGTGTGGACTCTGTCTCTCTCTATCTCTCTCGTTTTGGTAGTTTTCTTGCACTTTCAAGATTCAACTGAGCAGTTGTG
OsAPRL3 ..................................................----------------------------------------------------------------------------------------------------
OsAPRL3a ......................................................................................................................................................
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 TTATGTATCATTAAATGAGCTTGTATTTTCTTAGCTCGGGTGTGACATGAACATATAAGGTATTTTTGTTCCAAAATTCATGTCCTGGTCTTTGGACTTCAGAATTTCCTTTCCTTAAAAAATGTTTGGTAGTTATCTCTGTTTCTCTAT
OsAPRL3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL3a ..................................--------------------------------------------------------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 AAGTCATTTTCTAATGTTGAATTTATGTGATGAAGGTTTCGATGTTTCAATGACGTCAGAGGCCGTGCTACATTCAGTAGATGGTATTGAGCTTAAGAAGGATGCTGAACAGGAAAATTGTCCATTCTGGTGGGCACGCTCACCGGAGAA
OsAPRL3 -----------------------------------...................................................................................................................
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 AATACTTCAGCAGGATACTTATCTAGCACTGGCAACTGCATTTGTAATCTTGAGGTTACTGTATCTTCTCTTTCCAAAGATAGGTTCCTTCGCCAAACGGGCGTGGAGGAGGCATACTCTTTTTCCAAACTTGGTGGGTGTGCATGAATA
OsAPRL3 ......................................................................................................................................................
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 TTTCTTCACCTACCTTGAACAAGCAAGACACAAATTCTTTAGGTTATACCCTTCGAAGCGAGGGAATTTACAGGAAGGGGCCAGGAATGCCACTGCTTGGGCTTCCAAGTCATTAGCATCCGTCTCAATCGGAGAACCAAGCACTATTGG
OsAPRL3 ......................................................................................................................................................
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 AAGGACAAACTCTACAAATGAGCTAAGATAAAGTGCAACTTTTTTGTCCGCAGGGATATTTGTACAGTAGAGTATCAGGATATGAACACTAAGAATCTGTTGTAACCGGCTTTGTTAGATATTGTAACATCCTTAGAATAATCACTTTTA
OsAPRL3 ...............................-----------------------------------------------------------------------------------------------------------------------
OsAPRL3a ------------------------------------------------------------------------------------------------------------------------------------------------------
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os02g51850 CCATTTCCCATTGTTACTTTCACTTCGGTTTATAGCTTTGCCTCTTGCCTAAATTCATACTCTTGGGAGTATGTGTTGCAAAATATCTTTGTTGTGTGAAATGTGTACATGGTCTCTGCAATCCCAAGTTGATGACATTTGAATC
OsAPRL3 -------------------------------------------------------------------------------------------------------------------------------------------------
OsAPRL3a -------------------------------------------------------------------------------------------------------------------------------------------------
Exon III
Exon III
Intron III
Exon VI
297
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 ATCCCCCCTCTCTCGTCGTCGTCGTCGCCGCCGCCTCGCCGTCCCCCGCCATTGGAGCCATGGATCTGGCTCCCGGCCGCCGCGCGCGACTGCTGGTGGCGCTGGCCCTCGTCGTCCTCGTGGCCCTAGCCGCGCGATCCGGCGCCGAGG
OsPDIL5-1 -----------------------------------------------------------...........................................................................................
OsPDIL5-1a -----------------------------------------------------------...........................................................................................
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TCATCACCCTCACCGAGGAGACCTTCTCCGACAAGGTGTGCTCCTCTCTCCTCTCTCTCTCTCTCGTCTCCCGCCTTCCCCACCGGCGACGATGCTGTTTTGTTCTGTAGATCACTGAGGTTGATCGAGCTAGGAGAGTCCAGGTCAGTA
OsPDIL5-1 ...................................-------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ...................................-------------------------------------------------------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 CGGGCTTGCTTTGTTGTAGAATTATGTAATAGATTCCGGAGCACTTCGTACTATAGCGGCGATGCTTGGTTTCAGACCTGTAGCTGTTGTATTGCAGATCTATTGACAATTCAAGTTTGATGGGCGCCTGCGCGTCAGTGTTACTTCGCC
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TCTGGGATGGAATCATTGGAATGATGGATTATATGAGCGTCGGGGTTTTAATTATCCGGTTTCAATCACGGATTGGGAATTTAGAATCGGGTCACAGTGTAAACTTTTGTCTTGTTAGTTTGTGGAGAATAGATACCATTGCTTTCCAGG
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TTGCATGTTTGATACTAGTAAAAAAAAAGGGTCATAGAGAGCCACTGCTCGTGTTCCTATGTTTTTGCAAGTCGTTCACCGTTGTGGCGTGATGCTTGTATTTGTTCCAACTAAGCCAGTTTGATCATGAGCAATCCTCTCATTTTTAGA
OsPDIL5-1 -----------------------------------------------------------------------------------------------------------------------------------------------------.
OsPDIL5-1a -----------------------------------------------------------------------------------------------------------------------------------------------------.
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TAAAGGAGAAAGACACGGTATGGTTTGTGAAGTTCTGCGTTCCATGGTGCAAACACTGGTAAATCTCACTCTTAGCTCCATGATGAATATTGAATTACCGTATAAATCTTGTCATTAATGGCTTAAGCTAATTTGTTAGATGCTTGTCAT
OsPDIL5-1 ..........................................................--------------------------------------------------------------------------------------------
OsPDIL5-1a ..........................................................--------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TTGCAGTAAGAACCTTGGAACACTTTGGGAGGACTTGGGAAAGGTTATGGAAGGTGCGGATGAAATAGAGATAGGGCAAGTGGACTGTGGTGTTAGCAAACCAGTATGCTCAAAGGTGGATATCCATTCGTACCCCACATTCAAGGTGTT
OsPDIL5-1 ------................................................................................................................................................
OsPDIL5-1a ------................................................................................................................................................
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TTATGAAGGTGAAGAGGTTGCAAAATATAAAGGTAATCTTCGTTTATGATGCAAATTATTCTATCTTGCTATTGTGTGGAAAGGTCAGCTTAATCTAGTTTCAACTTTCTAGTCTACAATGAAAATTGAAAAAGAGAAAGGTATGTTTGG
OsPDIL5-1 ................................----------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a .................................................-----------------------------------------------------------------------------------------------------
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 ATCTGTTAGATTTTCTCCAATTCTCTTGAGGTTTAGGCCATGTGTTGGAAGCCATATCCCCCTGCAGTCTGAATTAAAGACTACAAACCATTTCAACTAATAGAATTGTGATAATGCCAAGCCCCACAAAAAAAAAAATCGAAGACCCTA
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
IExon I
Exon I
Intron I
Intron I
Exon II
Exon II
Intron II
298
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 GTGTTTGTTTCTCAAGTGCCATTTTTGGAGTAGCAAGTAGCATCTTCTGTGAAGATTCTGCCAAATCTGCTGATGAAAACATTTGATTATTTCTAGTATCTGCCAGCACTTGATTCTCCTCTTTATATTTTTCCGTTGCTTGAACTCGTT
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 ATATTGTTTTGTTTATGATTTTCTGCTCATGACATGACTTGTTATAGTTGAAGCATGCCAGGATACTGATTGTATCCTACAATTGTACGGCATGAAGAGCATACATTACCTCTAGCTCTAGGAAACTATGCATGTGCTCTAGTATCTCTG
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TCTTTTCCTGCCATTCCCTATCTTGTGAACATTGGAATGTTACTCATTCTGATACCTATCACTGCAATTCACAACTTAGAGCTTGATCTTCTGCTAACTGTTTGTACTTCAGAAAATATGCAAGTTTTTTTCTGACAGTTATGTGCGTAC
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 TAAAATGCAGATTGAATATATTGTCATCTTTTTCATCGCTGTTTCACCCTATCTTTTGTAGCTTTATGCTTTGTTACTGCAGCTTTGAGGGGCAACTTCTGTCTGTCCTTCATCTGACCACCCACCTGCTCGCTGATTTCTGTTGTTAAC
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 GTTAGATGCCTCCTAAAGGAAATCCTTGTGATCTTTTGTGCTACTGCAGGCCCAAGAAATGTGGAGTCACTGAAGAATTTTGTTTCGGATGAAGCCGAGAAAGCTGGGGAAGCCAAGCTTCAGGACAGTTGAACTGGAGCTCAAGTGTTC
OsPDIL5-1 -------------------------------------------------...................................................................................------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os03g17860 CATCCTACCACAGTTAAGCAACAATGTAGTACTGTTTGACATGCTTAAACTTTTTGATATAGTGAGCTTGTACTTCCTCCTGGCGTTTAGTAGAATGTTAACCACTGCATGAGTCGCATGAAACTATGTAATTTTCTGCTTCATACTGCA
OsPDIL5-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | .
Os03g17860 TTCAATTACAGCATGCTATACTATAAAGAAGAGTTCATGGAAAAAGCGCCTTTTTTTTTAAAAAAAAAGAAGAAGGCATCATAGAAGTTGCATTCCATGCC
OsPDIL5-1 -----------------------------------------------------------------------------------------------------
OsPDIL5-1a -----------------------------------------------------------------------------------------------------
Exon III
299
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 TCGTTAACCTCCATCTTCTCTTCTCCTCTCACGAGCTCGCAGCTCAAGCGCACCCAGCCATGGCGACTCCTCAGATCTCGCGCAAAGCCCTCGCCTCCCTCCTCCTCCTCGTCGCCGCCGCCGCCGCGGTCTCCACCGCGTCCGCCGACG
OsPDIL2-1a -----------------------------------------------------------...........................................................................................
OsPDIL2-1 -----------------------------------------------------------...........................................................................................
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 ACGTGCTCGCCCTCACCGAGTCCACCTTCGAGAAGGAGGTCGGCCAGGACCGCGCCGCCCTCGTCGAGTTCTACGCGCCATGGTAATAAGCGATGATGGTGTGCGTGCATCTTTGGGTTTTTCTTCTTCCTTTTCTTTTTCGGATCCGCG
OsPDIL2-1a ..................................................................................--------------------------------------------------------------------
OsPDIL2-1 ..................................................................................--------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 CGGGGCGTGTGTGTGTTCTGGATCTCTGTGATGATGAAATGGGCTCGGGATCGCGGCTGGATCGGTGGCGGCGTAGCTCTCATTCTCGGAATTTCGTAGCGGATTCTTGAGATCGGTGAGGTTTTGGGTGGCTTGTGTGGAGTGGATCGA
OsPDIL2-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 GTTGATGTTAGGCTGGAAAAATATTCGGATCTTTGGCTGAAGTATCGATCTTGGCCGCGGAGTGTTGCGTGGATCTTTCTTGACATCACAGATTCGTTTACTTTTCCGGTCCCTTATTTTGTAGTATCGATCAGATGTGTGAGATTTTGC
OsPDIL2-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 AAGGTTTTTAGGGCTTTTATGTTATATCTTGCGGTGGTTGGTGTTGGGGGAGGGTTTATGTATAGAGAAATTCGATATTTTTTAAATGTTTGTGATTGATTTTGTTTTCAGGTGTGGTCACTGCAAGAAGCTTGCCCCTGAATACGAAAA
OsPDIL2-1a ---------------------------------------------------------------------------------------------------------------.......................................
OsPDIL2-1 ---------------------------------------------------------------------------------------------------------------.......................................
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 ACTCGGTGCAAGTTTCAAGAAAGCCAAATCAGTCCTAATTGCGAAGGTAAATCTCATGTTTTTTAGGTGTTTGGAAATTTAATGTCAAGGCCGTTTTGTGTCATATCTTCGCTGTTTGCATGCCAGTAGTTCTCTATCGTTTGCATTCCA
OsPDIL2-1a ..............................................--------------------------------------------------------------------------------------------------------
OsPDIL2-1 ..............................................--------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 TATGGAAAGAAAATAAAAATGGCATTCCTGTTTAGTGGATATATGTTATTAGGATCTAATAATTTTATGTGTTTGTTGATGAATTGACTCCATTTTGTCTATCCAGGTTGACTGTGATGAGCACAAGAGTGTGTGCAGCAAGTATGGAGT
OsPDIL2-1a ----------------------------------------------------------------------------------------------------------............................................
OsPDIL2-1 ----------------------------------------------------------------------------------------------------------............................................
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 TTCTGGGTACCCCACAATCCAATGGTTCCCCAAAGGTTCCTTGGAGCCCAAGAAGTGAGAATACATGCTTCTTTGTTGATTACTCAATCACTACATTTTCTGTCGTAAAGAACAATGACCCTAATGTTAAACTTACTGTTTAGGTATGAA
OsPDIL2-1a ........................................................-----------------------------------------------------------------------------------------.....
OsPDIL2-1 ........................................................-----------------------------------------------------------------------------------------.....
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 GGTCAACGTACCGCGGAAGCCCTTGCAGAATATGTTAACTCTGAAGCAGGTAGACGAACGCACTTTCCAACCAAGCTATCGGTCTCCTTGTTTGGAAAATTAACTGCATTTTTTATTACCATGAAACATATTATGTATGATATTTCAGTT
OsPDIL2-1a .................................................-----------------------------------------------------------------------------------------------------
OsPDIL2-1 .................................................-----------------------------------------------------------------------------------------------------
JExon I
Exon I
Intron I
Intron I
Exon II
Exon II
Intron II
Intron II
Exon III
Exon III
Intron III
Intron III
Exon IV
Exon IV
Intron IV
Intron IV
300
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 TTCACCAGTCAGAGGATTATTTCATAAGCAATAAGTACTGAAATATCAATGCTACTTGGCTACTTCCTGCTATTATGGAGCCCAAAAGCTGTTGCAGCTAATTACATATCACTGCTTTCTCATTACATGTTGTCATGTCTCATTTTCATT
OsPDIL2-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 ATTTGTTGAACAGTTGGCTATTGGCAAACCAACATGCTATATTAGCTTTCAAAGTCCCAACACTGTTGTAAATCTTTCTGCCTTGTCTTTCAGCCACCAATGTAAAGATAGCTGCAGTTCCTTCTAGTGTCGTGGTTCTTACCCCAGAGA
OsPDIL2-1a ---------------------------------------------------------------------------------------------.........................................................
OsPDIL2-1 ---------------------------------------------------------------------------------------------.........................................................
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 CCTTCGACTCTGTTGTCCTTGATGAAACCAAAGATGTCCTTGTAGAGTTCTATGCCCCATGGTATGCTATTATGATCAGCCTGTTGTTCATCAAACCTTTTTATCATGCTGTATCAATGAATTTAATTTTGTTGGATGCTGCTTTGTTTT
OsPDIL2-1a ...............................................................---------------------------------------------------------------------------------------
OsPDIL2-1 ...............................................................---------------------------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 GCAGGTGTGGTCACTGCAAGCATCTTGCTCCGGTTAGTAGTACACCTCAGTATTTTGCAAAGTTGCAAAATAATGCATTGTCATTTTTCTATTTTGTCTAAACTCAAACTTGCACTTGTTTTAGATTTATGAGAAGCTAGCTTCTGTTTA
OsPDIL2-1a ------..........................--------------------------------------------------------------------------------------------..........................
OsPDIL2-1 ------..........................--------------------------------------------------------------------------------------------..........................
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 TAAGCAAGATGAGGGTGTTGTTATTGCCAATCTTGATGCTGACAAACACACTGCCTTGGCAGAGAAGTATGCTTTCTGAACTTTGTTCTAATAGTTCTTGTCTACATTGATTACCCTCGACATTTCGATGTAACGGCATAGCTTATGTTG
OsPDIL2-1a .......................................................................-------------------------------------------------------------------------------
OsPDIL2-1 .......................................................................-------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 GAATGGTATTTGCTTTCCTGAAGAAAATTTTCTACTGTTATTGTACTTCTCCTAACTGTTAGATCTAACATACACATACCTGTTTTTATAGGATGTGTGGTTGGTCATGTATGTTTTTCTTCCCCATAAGAGAATTTTTAATCATGCATG
OsPDIL2-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 CCACTTCAGGTATGGAGTTTCTGGTTTCCCCACATTGAAATTCTTCCCAAAGGGAAACAAAGCTGGTGAAGATTATGATGGCGGTCGCGAGTTGGATGACTTCGTCAAGTTTATTAATGAGAAATGTGGAACCAGCCGTGATTCAAAGGG
OsPDIL2-1a --------------........................................................................................................................................
OsPDIL2-1 --------------........................................................................................................................................
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 TCAGCTTACTTCAGAGGTAGTGCTTTTATATAGAAGGTACTTAAATACTTAAATTTACATAATCCAATTCCAATATAATCTATTGGTTGCTTCTGTAGGCTGGCATTGTCGAAAGCTTGGCTCCTCTTGTGAAGGAGTTCCTTGGTGCAG
OsPDIL2-1a ................----------------------------------------------------------------------------------....................................................
OsPDIL2-1 ................----------------------------------------------------------------------------------....................................................
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 CAAATGATAAGCGGAAAGAAGCCCTCTCCAAAATGGAAGAGGATGTTGCGAAGCTCACTGGCCCTGCTGCCAAGTACTAAATTTTCATCTCTGTTTTTAAATTTTGTTATGTAGGTTTGGCATCGTCACTGTGTGAATTTAACCATTTTG
OsPDIL2-1a .........................................................................-----------------------------------------------------------------------------
OsPDIL2-1 .............................................................................-------------------------------------------------------------------------
Exon V
Exon V
Intron V
Intron V
Exon VI
Exon VI
Intron VI
Intron VI
Exon VII
Exon VII
Intron VII
Intron VII
Exon VIII
Exon VIII
Intron VIII
Intron VIII
Exon IX
Exon IX
Intron IX
Intron IX
301
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 CTAAAGCAGGTACGGAAAGATCTATGTGAACTCTGCAAAGAAGATCATGGAGAAGGGCTCTGAATACACTAAGAAGGAATCTGAGAGGCTTCAACGCATGTTGGAGAAGGTGAGCAACAAAAGTTTCTATTCCTCAATTCACTTTGGACA
OsPDIL2-1a ------.......................................................................................................-----------------------------------------
OsPDIL2-1 -------------................................................................................................-----------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 TCATATTACTTAATCTAGAATCTGATTTCCATGCTTCATCATATGACAGTCGATCAGTCCTTCCAAAGCCGATGAATTCGTCATCAAGAAGAACATCCTTTCGACTTTCTCCTCTTAAAGGGTGTCCCACGTACCCCGACGAGCAATTGG
OsPDIL2-1a -------------------------------------------------.....................................................................--------------------------------
OsPDIL2-1 -------------------------------------------------.....................................................................--------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 GGTATAGTCAGTAGCGCAATGCCTGTCTCCAAGAATGGAAAACAGAAAGAAATGAAAAGAAAAGGCAATATCGATATCAGCAAGAGATGGCAATGGAAGTTCGTTAGTTGGGTATTCTGTTGAACAAACATGTATCCCACTGCCCGGCAT
OsPDIL2-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 GATCTTTTTTTTTTCTTTCTGCTATTTGCTTTTGTGGTTACAGGGATTGACATAGCAATCAGCTAGTTTACTCAAGAACACAAGAGGTTTTGTAACCATCCCGTGATGAGTGATGACACCGTTCTACGTTTGTCAATCCAATAATTTGAT
OsPDIL2-1a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-1 ------------------------------------------------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g06430 AATTCAGTTTACATTATTTCTGTTTACCCTGGTGAAAGAGAGCATATGTTGCTTTTGCTT
OsPDIL2-1a ------------------------------------------------------------
OsPDIL2-1 ------------------------------------------------------------
Exon X
Exon X
Intron X
Intron X
Exon XI
Exon XI
302
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 CTAGCCTAGTCCCACCACTAGGACTCAACGAGAAGGGGGGCAGGTGTCCGAAGCGGATCACGGCGGCGGGAGAGAGATCCGGTGGCCCGATCCATGGCTGCGGCGGCGGTGGCGCGCCGCGTCGTCCTGGTGCTCGTCCTCGCCGCCGCC
OsQSOXL1 ---------------------------------------------------------------------------------------------.........................................................
OsQSOXL1a ---------------------------------------------------------------------------------------------.........................................................
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TCCCTCGCCGCGGCGCCGCGCGGGGCGGCCGCTCGCTCGCTCGGCGGCAGGGAGGGCCCCGGGGAGGTCGACGCCGACGCCGCCGTCGACCTCAACGCCACCAACTTCGACGCCTTCCTCAAGGCCTCGCTGGAGCCCTGGGCCGTCGTC
OsQSOXL1 ......................................................................................................................................................
OsQSOXL1a ......................................................................................................................................................
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GAGTTCTTCGCCCACTGGTTCGACTCTCGCCTTTGACTCGTTTTTTTCTCCCCGATTCGTGACTGAGGCGTTAGTTGTAGTACTTGGTAGAATCGATAAGTGGATGAGAATCGTGGTAGTCCGATGGCGTGAGATGCGACTGCGGGGTTT
OsQSOXL1 .................-------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a .................-------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 CGTCGTCTTCCCCGATGTATGGTTTATAGAATCGAAATGATTTACTCGTTTCGTCGTCTGTGAGCAAGTGAATGCCAATATAATCCACAATTTTTGGGGTTTCAAGCATGTAGGGTTGTGTAGGTGTTGCAAGGTCTGATTGGTGGGATG
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GATGCGTGTTGTTTTTTTTTTTTTTGAACGAATCGGCAAGCTGCCAATTTCATTGAATAGAGAAGGAGTAAGGATGCGTGTTGTTCAAAACTCGATGAGAGTAACCAATAAATGCTTCGCTCGTACTACCTTGAAATGTCATGTGAGGAT
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GCGACCCTGGGAGTGTTGTCCGTGTTGTAGTACTGTGACCTGTGAAGTTGTGAGCTAGAAGAAAGGTGCAGACATGAATGAGCCCCGAAGCTTGGAAATTTGTTATATGTAGGACTAAATCGGATGTAGGCCAGCAGTTAATTATGGAAA
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 ATCAGTGGAGCATTTACTGTAATGATGAAAGAGGAATTGACTGGGTTGTAAGTTTATCACTATATAGTTTCTCAGTGTTGCCACTTACTCAGAGTATTGCTTCGATGCGATATTTCTTGTGTGATTTGGGGTGTGCGGAACTTTCTCAAA
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 AGCATAAGTACGGTCATTTTTGCTCTATGTAAGTTCCACTGTTCCCCACATTGGGCTATAAATGCCATGCCGTTTAATTCTAATTTGCAGAGATCATCTCTTTTTGAAATTTTAGCTTCATGAGATTGAGTTATATTTAAATGCTTCTTT
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TGGATGTTAACAGGAAAAATAATACACTTATATGAGCATTGATACTGTACCTCACATGATTTAATGGAACTTGACGATTTGATTAATACACATGGACCTGTTTATGGGGAATCAAGGACTTGAATGATTAGCATCCTTTTGTTATCCTTT
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
KExon I
Exon I
Intron I
Intron I
303
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TCACTCTAATGTCCATGTTCTGGTCTTCCAAATATTCCATGTCAAGTTATTTTGATATTATATTCCATTGGTTAATCATTATAAGTCGTCACTATTCAGAATAGGTTAGTCCATATGCATTAGATATGAACTATGAAGTGGCGTATAGTT
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TCTCCAGAGCCTCAAGTTTTCACTTACTCGAGATAATTTTAATTGGAATGAACTTCTATTGGTCATATCGGTATTCTTTTTTTTTTCTTTTGAGGCTGCCTAGATGGCATTTGTTTTATTGTTGAATTGATGCTAATTGAAATACGTACC
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GTGTTTGCTCAGGTGTCCAGCTTGCAGAAACTACAAGGTATGAAGCTTATTGTGGTAGCTGTGTACTTAATTTAAAAAACAGTGGTGCACAGTGTATTTATGAACAGCAAATTGACTCTTGCTAAATTCCTTCCTTACATCCAGCCTCAT
OsQSOXL1 ------------.........................-----------------------------------------------------------------------------------------------------------......
OsQSOXL1a ------------.........................-----------------------------------------------------------------------------------------------------------......
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TATGAGAAGGTTGCAAAACTATTCAATGGTCGGGACGCTGCACATCCAGGGTTAATACTGATGGCTAGGGTTGATTGTGCATCAAAGGTATAGTAGCTCTGGTGAAGAGTGATAACTTTCGTCAGTCTCCTAAATGTAGACAACTAAAGC
OsQSOXL1 .......................................................................................---------------------------------------------------------------
OsQSOXL1a .......................................................................................---------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 ACTTACAAAAGGGATTAGGTTTGATGGAATGGTCCAGTTAAATCCTTAACTGGATGATGAGATTATCCACAGGAGTATATCTTCAATCCTTTGATATCTATTTTTCGTTACCAAACTGAAAGCTATATTGCAATGAAGCTTTGGCACCTC
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TTATTTTGACTGATTTTAGCTTCCATGTTTTGTCAAAATGTCCAATAATTTGGTAATTTCAATGGAGTAATACTTTATTTCTTTTGATTGTTTGACAGCACAGGTGAACATCGATCTTTGCAATAGATTCTCAGTTGACCATTACCCTTT
OsQSOXL1 -------------------------------------------------------------------------------------------------------...............................................
OsQSOXL1a -------------------------------------------------------------------------------------------------------...............................................
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 CCTACTTTGGGGTCCACCAACAAAATTTGCTTCTGCTAAGTGGGATCCCAAGCAAGAGAATAATGAAATAAAGTTAATTGATGATGGAAGAACAGCAGAACGTTTACTGAAGTGGATAAATAATCAGATGAAAAGGCAAGTTATTATCCT
OsQSOXL1 .......................................................................................................................................---------------
OsQSOXL1a .......................................................................................................................................---------------
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 AGTCTTTCCTGCTCTACCTAAAAGTTCAGTAGATATGGAAACAGGAGCTACCTGTACTATGCTCATGCCTCTCTCTATTGACTGTTACCTTTTTGGTTTGTTTGACCATTATAGGCATGTTGGTATTTTATTGATGCATTGTTTATTTAT
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GGTATCTGCAGCTCTTTCAGTTTAGAAGACAAAAAGTACGAGAATGAAAATATGCTTCCAAAGAATGCTTCGGATCCTGAGCAGGTTCAATTATCAGATATTTCTTTGAAACAATTTGCTTGAGATACTGCTATGTGCACTATGTTTCAA
OsQSOXL1 -----------.........................................................................------------------------------------------------------------------
OsQSOXL1a -----------.........................................................................------------------------------------------------------------------
Exon II
Exon II
Intron II
Intron II
Exon III
Exon III
Intron III
Intron III
Exon IV
Exon IV
Intron IV
Intron IV
Exon V
Exon V
Intron V
Intron V
304
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TTTTCTTTAACGTTTGTTGACTTAATTCGATCTTTTGTTTTAGATTGTTCAAGCAATTTACGATGTTGAGGAAGCAACAGCTCAAGCGTTACAGATAATTTTGGAGCGCAAGGTGAGTTAACGATGAAATTTCACAGCAGATGTGATGTT
OsQSOXL1 -------------------------------------------.....................................................................--------------------------------------
OsQSOXL1a -------------------------------------------.....................................................................--------------------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TGCTCTTCTTTTTCTGACTATGGCCTAATAATGTTACTTGCAAACTGTAGACGATCAAACCAAAGAATCGTGATTCGCTCATCAGATTTTTACAAATTTTGGTGGCTCGTCACCCATCCAAGAGGTACGAGTGAACCTTTAAATTCCTAG
OsQSOXL1 --------------------------------------------------..........................................................................--------------------------
OsQSOXL1a --------------------------------------------------..........................................................................--------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TAGAGAAAATGCATATAACTTTCTTTGTGCATTTGGTTGATTCTTTAGGTGCCGAAGGGGATCTGCTGAGCTACTTATTAACTTCGATGATCACTGGTCATCGAATCTGTCGTTAAGTTCACAAGAGGGTTCTAAATTGTTGGAAAGTGT
OsQSOXL1 ------------------------------------------------......................................................................................................
OsQSOXL1a ------------------------------------------------......................................................................................................
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TGCGGAAGAGAACCACTGGATCTGTGGAAAAGAGGTGCCACGTGGATATTGGGTATGTTGTCTAATGCTAGGCCTAGCGGTTGGCAATGAATTCTTCATTAGGCATTCTTTTATACTAAACTAGAAAATGCAACTTCATATGAATCATTC
OsQSOXL1 ...................................................---------------------------------------------------------------------------------------------------
OsQSOXL1a ...................................................---------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TTAATTTTGATTTTGAGATATTCTCATTAAATGTTGCAGTAAATCCGTTATATGAATGATAGCATTAGGAGCAAGCAACATCTTGTATCTTTTGACATAGTTTACTTCAACAGCAGCATTATTTGCATGTAGCTTCTTTAGTACTGAGTT
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 AATCTAGTTTCATTGCAGACAGAGGATCCTTAACCACTGTAGAATGTTTAGTCGACACACGTGATCCACATATTTTAGATCCCACAGCCCATATTTATTTACATATCTTGATCTTATCACAAGTAATATCTGTACCTAGCAATAGTTATT
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 CCCTTGAATTTTCTCTTCTTATTTCTGTGTGCCAATGTTGCAGCTGTTCTGCCGCGGCAGTAAAAGTGAAACAAGAGGATTTAGGTGACGCCTCTCTCTCTCTGCAAATGCTTCGAACATATATCTTTACAGCACCTTTACTGAAAGGCC
OsQSOXL1 ------------------------------------------..........................................------------------------------------------------------------------
OsQSOXL1a ------------------------------------------..........................................------------------------------------------------------------------
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 ATAGGAGTTGATAAACTATAACACACATACATCTTTTTTTATTGTATCAGCTGTGGTCTATGGGTTTTGATGCATTCACTAACCGTCCGAATTGGGGATGGAGAGAGCCAGTCAACCTTTACATCAATATGTGATTTTATTCACAACTTC
OsQSOXL1 --------------------------------------------------....................................................................................................
OsQSOXL1a --------------------------------------------------....................................................................................................
3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TTCATCTGTGAGGAATGCCGCAAGCACTTTTATGAAATGTGTTCAAGGTTTGGAAAACTAACTTTGTGTTGTACCATCATTCAAAAAATATATCTACGACATGCCTGACTGTAGCCATTTACCTGTAGCGTGTCAGCCCCCTTCAGAACT
OsQSOXL1 ...............................................---------------------------------------------------------------------------------......................
OsQSOXL1a ...............................................---------------------------------------------------------------------------------......................
Exon VI
Exon VI
Intron VI
Intron VI
Exon VII
Exon VII
Intron VII
Intron VII
Exon VIII
Exon VIII
Intron VIII
Intron VIII
Exon IX
Exon IX
Intron IX
Intron IX
Exon X
Exon X
Intron X
Intron X
Exon XI
Exon XI
305
4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GCTCGTGAGCTCAGTCTCTGGTTATGGAGCACACATAACAAAGTCAATATGAGATTGATGAAAGAAGAAAAGGATATGGGAACTGGTGATCCCTTGTTTCCGAAGGTTACCTGGCCTCCAAATCAGCTCTGCCCATCTTGCTACCGCTCA
OsQSOXL1 ......................................................................................................................................................
OsQSOXL1a ......................................................................................................................................................
4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 AGCAAGGTCACCGATGGAGCTGTGGACTGGAACGAGGATGCAGTGTATCAATTCTTGGTTAACTACTATGGAAAGAAGCTTGTATCATCCTACAAGGAGACCTACATGGAGTCTCTTCAGCAACAAGAGAAGAAGATAGTTTCAGAGGAT
OsQSOXL1 ......................................................................................................................................................
OsQSOXL1a ......................................................................................................................................................
4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TCATCGATTTCTAATGCTGCAAGCGTCCCAATTGGAGCTGCCCTGGGTGTTGCGATTGCCAGCTGTACATTTGGGGCGCTGGCTTGCTTCTGGAGGGCCCAGCAGAAGAACAGAAAGTACTCACACCATTTACGCTCTTTAAAGAAAATA
OsQSOXL1 ....................................................................................................................----------------------------------
OsQSOXL1a ......................................................................................................................................................
4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 TGATTAGGAAAAAACATTTTAGGTTGGTAGGGTACCATTGTTACCGCGTGACTACGTGGAATTTCATATCACAGAGAAGTGATTAGGTTCTATTTGTCAATTTTTTTACAGGCAAAGAAAGAACTGGAACTGAAAAATAGCCTACCAGTT
OsQSOXL1 ---------------------------------------------------------------------------------------------------------------......................-----------------
OsQSOXL1a ...---------------------------------------------------------------------------------------------------------------------------------------------------
4660 4670 4680 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GCTCACCCTGCACTTCATCAGAGGGGCTCATCAATGAATGTGTATTTTTCTGGACGGATTAAAATCTTGGTAAGTGCTCCAAACAGTGTGGGAAAGGAAATGGTCGGGCAGTCAGTTACAGATGAAAAACTCACAGAAGTACGATGATTA
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
4810 4820 4830 4840 4850 4860 4870 4880 4890 4900 4910 4920 4930 4940 4950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 ACTCAAGTGTATTTTTTGTGCAACGATGTGAGCTTTGTTGCATCATGTCATAGATCAACGTGTAATAGTTTGACATATAGATTTCGGATGATTGGTGTATACGTTCGGATGTTACTATCCATTTAACGAGTGAAATTTTGAATTCAGTTC
OsQSOXL1 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsQSOXL1a ------------------------------------------------------------------------------------------------------------------------------------------------------
4960 4970 4980 4990 5000 5010 5020
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os05g47930 GTAGCTTTCTACTAACCTTTATCGTTCTCAGATATTTACCTGCAATTAAATGCTTTGTGATCGATTTATG
OsQSOXL1 ----------------------------------------------------------------------
OsQSOXL1a ----------------------------------------------------------------------
Intron XI
Exon XII
306
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 ATTGCAGTTGCAGTTACAACCGGCAAATCTCTCGCGACCCTGCACCGTAGCAGCGTCGACGCGAGGCGGACAGCCTCTTCTTCCTCCTCCCTTTCCACCCCCCACAGAGATCTCGCCTCTCTCCTCCTCCCTTTCCCTCTCCTTCCTTCC
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AGATCTCTCCACGCGCCGCCCCGATCTCTTCCCCCCACCCCCACCTCGTCACCGGCGGGGAGGAGGGCCCGGCCGCCGGCAGCCATGATCTCCTCCAGTAAGCTCAAGTCCGTCGACTTCTACAGGTCCCCTCCACTCCCGATCTCTCTC
OsPDIL5-4 ------------------------------------------------------------------------------------.........................................-------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TCTCTCTCCCTCCATTGCTTGCTATGGTACTCCCTGTGCGCGCGCGCGCGGGGGGGATTTCGACGCGGTCCTCGGCTGGGGGTGCTGGTCCGGGTTCGGATTGGGTGAATTCTCTTGCCTGGCTGTTCTGGCGATTGCAGTAGATGGTTT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 GTGATGGTTCGTTGATGCACCGAGAAAATCGGGGTTGCGTGGGCGTATGTGGTGGTATTGACCATTCGTGCGGTCGTTGCGAGTAGTGTAATGTCTGGTGTATTTTCTACTGAGATGGGTAATTTGGTTGATGCGCTTTTTCTTTTCGTG
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CTTGCAGGAAAATTCCTAGGGATTTGACCGAGGCATCACTATCTGGGGCTGGATTGTCCATTGTAGCAGCACTAGCTATGGTGTTTTTGTTTGGAATGGTCAGTTTTCGTTGGATTAGCGATTGCACTGTTATTTTTAGATTTCATTTTT
OsPDIL5-4 -------...........................................................................................----------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TCACTCTGGTTCCAGTCTTTAGGGAAACAAACAAAAAATGGTTGGACTGTTCTTCTGTTCCATTTGGTGATTGTAGACTGAAATTTGGTACAGAGCCATACTAAAATGATAGAGCATTTATTTATTTATATGCAGATTAGTTGTCTTCTA
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CCATTCCAACTGCTGTAGTGTAGCTAATCGCTATCGCCGGATCAATGTAGAGTCTCGATTTGCTTGTTTCATTTGATGATTGAGAATAGCTCGTTTTTCCTGATATCTGTTTCAATTGACTATTAGAGCTTCATGATAGGCTAGATTCTT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AACTGAACCTATTTTGGTTGAATATTTTTGACCGAATTATCAATTGGTCCATCCAACCTTGAAAGTGGCAGTTAAGTAATGTATATGCCCAAGTATATTGTAATTACTCCGGAACATATGCATAATTTTGGCTGTTGCTTGTTAAGGCTT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 GCAATGTGCTATACTTTTCACTATGCTGAGCTGATTCCTTAATCAAGCTTTAGACTAGTTTAAAGGTTCATTTCAACTGAAAAGCTAAATCTGTTCAATTGTTATTGCAGGAACTGAGTAATTACTTAGCAGTCAATACTAGCACATCTG
OsPDIL5-4 --------------------------------------------------------------------------------------------------------------........................................
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon I Intron I
Exon II
L
307
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TGATTGTTGATAGGAGTTCAGATGGGGAGTTCTTACGGATAGATTTTAACTTAAGGTAAATTCTATGGCACATGCTGCACATCTCAACAAGTAATAAGGTTAGAATACAAAATGAGAACCTTCATGATCACATAGATAATACTGCTATCA
OsPDIL5-4 .......................................................-----------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TTATGGTTCATAGAACTAATGTCTTCCTAATTCTCTAATCTGCTGATAGTAATTTTATCCCTATTGTGATTAATTTTAATGGAATACTATATATGCAACAGCAAAGCACAGCGTTAATTAATGTCAGGCACTCAGTACATTCTTTTCAAT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 GTATTTGTACTCATTTGGTGCATTGTGGAAAAAAACATATGATTCTCAGTTAATTCATTTATCTTCAAAGGTCACCTAATTACCAGATCCAACCATTTGTAGTAATATGTTTGTAGAATAATTCTGATGTTGTTCTTTTACTCAACACTT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TCAACCCTTGGTCTATATGAACCTGAGAGCCATTGTTTATACTTGAAATTGATGCTTTCCAGCAAACAAGGACTAGTCAGGCACTGCTCATTTATTTGTATGTGATAATGCATAGGAGGGTATCTACATTAGTCTAGCTGGTTGCCCATT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 GTTTTCTATGGAATGACATCAGCAAAGCTGGACTCAACTTTTCCTTGTTTTTCCAGTAGATGCTCACGATCTGGTTCTGTGATTTACAGCTTTCCTGCACTTTCATGCGAATTTGCATCAGTTGATGTCAGTGATGTTCTGGGAACAGTA
OsPDIL5-4 -----------------------------------------------------------------------------------------..........................................................---
OsPDIL5-4a ----------------------------------------------------------------------------------------------------------------------------.......................---
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AGATTCTAACTTGACCTCATGTACTCTGTTTTTGTTCATCTGCCGTCTAGAATGAGGATATCATTGAAGGACATGAAGCTCATCTTGCGCGTTCCATAAAAACTATTGTTGACTTTATTTTCTTGTTAACTTGCCATTACAAATAACTTT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CCACTAGCTTTTAAAAGTACTCCCTCCGTTTCACAATGTAAGTCATTCTATCATTTCCCATATTCATATTGATGTTAATGAATCTAGACATATATATCTATCTAGATTCATTAACAACAATATGAATGTGGAAAATGCTAGAATGACTTA
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CATTGTGAAACGGAGGAAGTAAGCTCTACATGTATATTTTACACCAATGTTAAAAATACGAAATATGACAATTTATCAGTTTTTGTAAAGTTCTGTGCATGTCAAAAGAATGAAACCTATCCTAAATTCCTAACTAACTTTCTATCTATA
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AGCATTTCTTGATCATATCTTGCCTTTTGTAGCTCGAGTTTTTCTCCATGCTGGTTTAACCTTAAAGTTTCTGTTCAAAACACATGCAGAACAGATTGAACATAACGAAAACTGTTCGCAAATATTCAATTGATCGGAATTTAGTGCCTA
OsPDIL5-4 -----------------------------------------------------------------------------------------.............................................................
OsPDIL5-4a ----------------------------------------------------------------------------------------------........................................................
Intron II
Exon III
Exon I
Intron IIIIntron I
Exon IV
Exon II
308
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CTGGATCCGAGTTCCACCCAGGACCTATTCCTACGGTCAGCAAACATGGAGATGATGTTGAAGAAAACCATGATGATGGTTCAGTTCCCTTGTCCTCTCGCAATTTTGATAGCTATTCACACCAGTAAGTATTCAAATATGTATTTCAAT
OsPDIL5-4 .............................................................................................................................-------------------------
OsPDIL5-4a .............................................................................................................................-------------------------
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 ATCCACCTATCCGTAACACATAACATATAGCATTTTAGCAATTGTGCGTTTCAATACCATATCAACGGTCAACCTATTATTCATGTATGCTGTCTTCTTTCTCCTTCACATTCATGGAGAAATGTACTTTATATTCATAGCTCCAATCAC
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TTCAACCAGATTGTCTATGGCAAATTGGTGATAATCTTCACTTTTACCTGCCTCAAGTATTTTTCCTTGAAGTGCCGTTCGGTATATCTAGTTAATCCCTCTTGTATTGTTTCGGAGCCCAAATATTGTTTCCACTGATTATTATCATGC
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 ACTTCCTTTTCAGGTATCCTGTTTTGGTTGTTAATTTTTATGCCCCCTGGTGTTACTGGAGCAATCGATTGGTTAGAATTCATCTTGTTCCTTTGCATTTAGTGATTTTCTAGTATATGCTACTCTATCTGTAAGGAAATGATTAGTAAA
OsPDIL5-4 --------------.........................................................-------------------------------------------------------------------------------
OsPDIL5-4a --------------.........................................................-------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TTATTGCTGGTAAAGTAATGTAAGTATGGCACGGCTTATTGAACTAACAAATGGAGACATTTCTAATTACTTGATCTTCCTCCAGAAACCTTCGTGGGAGAAGACTGCAAAAATAATGAGGGAGAGGTACATACTGTTTCTCTTTGTTTG
OsPDIL5-4 -------------------------------------------------------------------------------------.........................................------------------------
OsPDIL5-4a -------------------------------------------------------------------------------------.........................................------------------------
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TGTTTTGTGGTTTTAATGTGCTGTTTAATCTATACCATGTTGTTTTTTTTTTCTGCAGATATGATCCTGAAATGGATGGCAGAATCATTCTTGCCAAGGTTGACTGCACTGAAGAAATTGACTTGTGTAGGAGGTTCGTCTGTACTTAAT
OsPDIL5-4 ----------------------------------------------------------...........................................................................-----------------
OsPDIL5-4a ----------------------------------------------------------...........................................................................-----------------
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TATTATTCATATCCAAAACAAGGACAAAGCATATTTAGGCTGCAACATGAACTTTACAAATCCCTTTTTGATCCATGTACCAAGAGAAATTTAGTTAATTAGTTTTTAGAGTAAAGTCCATCACCGGTCCCTAAACTTGTACCGCTGTGT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CATCCCGGTCCCTAAACTCGCAAATCGATCATTCAGGTCCTTAAACTTGTTCGACTGTATCATCCCGGTCCCTAAACTTGCAGATCACTCGTTTAGGTCCTCCAACTTGTTCAGTTGTGTCACCCCGGTCCCTAAACAGGACGGTCTAAA
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AACTTTATACCAAAAAATAATTCATAACTTTTTCATGTGAACTCTAATAAAGACAAACTTTATATCAAAATTGTAGCCCTCGACGCGACCTACAACTTTGTAGTTGAAAATTTTTTGAATTAAAACTGTTTAGGGTCCCAAAATATTGTT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
Intron IV
Intron II
Exon V
Exon III
Intron V
Intron III
Exon VI
Exon IV
Intron VI
Intron IV
Exon VIIExon V
Intron VIIIntron V
309
4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 GCATGTTTATAGATTTTGTAATTTTAATTTTAAAATTACATTTTGGGACAAAAAATGACTTAAAATAAAAAAATTATCAACTACAAAGTTGTAGATCGCGTCGAGGGCTACAAGTTTGATATAAAGTTTGTTTTCATTAGAGTTCACATG
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AAAAAGTTATGAATTATTTTTAAATATAAAGTCTTTAGACCGTCCTATTTGACCCAGATGATATTCAAATCCAAGTTTAGGGACTGGGGTGACACAACTGAACAAATTGGAGGACCTATACGAGTGATCTACAAGTTTAGGGACCGGGAT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 GACACAGTCGAACAAGTTTGAGGACCTGAACGGTCGATTTGCGAGTTTAGGGACCGGGATGACACAACGATACAAGTTTAGGGACCGGTGATGGACTTTACTCTAGTTTTTAATATCCCCAACAAAGTGTCATTATTATGCCACATCTTT
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CATGAGCTATTCGTGTTTGTGTCCTCTTATCCTGGTTCAGTGGATGCTTATGCCTTCTGGACTTGTTTGCATTTGGAATGATTCAAAAGCCTTTATTCCATAGATCACAACCCTAATTGTTACTGACCATGTTTTTATAAGAAAATGAGC
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
4660 4670 4680 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CATTCTGCTTCTGATGACACTGGTTTCTGTGTAATAGTGCCATTGCCATTGATGCACATAGTAACATTTCAAGCTATGCGCATTGGATGTGAACCACAACTCCTCAATAGAAGCCTTAAATTGTTCTCCCACTAGTTTTACCTAATTATA
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
4810 4820 4830 4840 4850 4860 4870 4880 4890 4900 4910 4920 4930 4940 4950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TGTGCTCTTGTTATGCTTGTTCGTCATTAAATTAAGGACTACAGTTCATTGTTTTAATTTATTAAATGCAGGCACCATATACAAGGTTATCCATCCATTCGCATTTTCCGTAAAGGGAGTGATCTTAAGTAAGTTCAAGTAAATCTTACA
OsPDIL5-4 -----------------------------------------------------------------------.........................................................----------------------
OsPDIL5-4a -----------------------------------------------------------------------.........................................................----------------------
4960 4970 4980 4990 5000 5010 5020 5030 5040 5050 5060 5070 5080 5090 5100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 GTCTTCCGGATACTCTACTGAGAGTTAACCTGTGGCGCAAGACTTGTATTATTATTATTTTTTTGCTCATGTTCCAGAAACAAACAAATTGACATTTGTGCAGGGAAAACCAGGGTCACCATGATCATGAATCATACTACGGCGATCGTG
OsPDIL5-4 -------------------------------------------------------------------------------------------------------...............................................
OsPDIL5-4a -------------------------------------------------------------------------------------------------------...............................................
5110 5120 5130 5140 5150 5160 5170 5180 5190 5200 5210 5220 5230 5240 5250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 ATACTGAGAGTTTAGTAGCGGTATGTATATTCTGTTACCCACTTATTTTTCTTGATGTACTGCATGATATATTCTTCACCCTAAGCTTTAGCTTACCTGTAAAAGGTATTGTCCCCATTTTCCTAGGGAGAACAAAAGGCTAACTATCCT
OsPDIL5-4 ....................----------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ....................----------------------------------------------------------------------------------------------------------------------------------
5260 5270 5280 5290 5300 5310 5320 5330 5340 5350 5360 5370 5380 5390 5400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TTGCTTGTAAATCACTGACTTTGTTCATGGTCTTGTCAAATTGTGGATAAAATTCCTGACATTTTTTTGGGCCTCAGGCAATGGAAACTTATGTTGCAAACATCCCAAAAGATGCCCATGTTCTAGCTTTGGAGGACAAATCCAATAAGA
OsPDIL5-4 -----------------------------------------------------------------------------.........................................................................
OsPDIL5-4a -----------------------------------------------------------------------------.........................................................................
Exon VIIIExon VI
Intron VIIIIntron VI
Exon IX
Exon VII
Intron IX
Intron VII
Exon X
Exon VIII
310
5410 5420 5430 5440 5450 5460 5470 5480 5490 5500 5510 5520 5530 5540 5550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CTGTTGATCCTGCAAAACGCCCTGCTCCATTGACCAGTGGATGCAGAATAGAAGGTTTTGTGCGGGTAAAAAAGGTAAATGAACTTCTGAGGTGTAATAGTTGTAGTGTTGATGATGATCTCTATCTGTGGTATTTGATTTCTGGTTCCT
OsPDIL5-4 ..........................................................................----------------------------------------------------------------------------
OsPDIL5-4a ..........................................................................----------------------------------------------------------------------------
5560 5570 5580 5590 5600 5610 5620 5630 5640 5650 5660 5670 5680 5690 5700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CTTTATCAGGTCCCTGGGAGTGTTGTAATCTCAGCTCGATCTGGTTCCCACTCCTTTGATCCATCCCAGATAAATGTTTCCCACTATGTAACACAGTTCTCTTTTGGCAAAAGGCTATCGGCAAAGATGTTTAATGAACTGAAAAGACTA
OsPDIL5-4 ---------.............................................................................................................................................
OsPDIL5-4a ---------.............................................................................................................................................
5710 5720 5730 5740 5750 5760 5770 5780 5790 5800 5810 5820 5830 5840 5850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 ACTCCCTATGTTGGTGGCCACCATGATAGATTAGCTGGTCAATCCTACATTGTTAAGCATGGTGATGTCAATGCCAACGTTACTGTAAGTCCCTAAATTTTGCTTTGTTTTTGTTATCAAGATGATTACAGCAGGCATGCCTTCCAGCAC
OsPDIL5-4 ....................................................................................------------------------------------------------------------------
OsPDIL5-4a ....................................................................................------------------------------------------------------------------
5860 5870 5880 5890 5900 5910 5920 5930 5940 5950 5960 5970 5980 5990 6000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TAAATTTTCTGGTTTCTTATGGAGTAACATAGTTTTTGTTTTTTGTCATTCTTAGATTGAGCATTACCTGCAAATTGTGAAAACTGAGCTTGTTACACTGAGATCATCAAAGGAATTGAAACTGGTTGAGGAGTATGAATACACAGCGCA
OsPDIL5-4 -------------------------------------------------------...............................................................................................
OsPDIL5-4a -------------------------------------------------------...............................................................................................
6010 6020 6030 6040 6050 6060 6070 6080 6090 6100 6110 6120 6130 6140 6150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 CAGCAGCTTGGTGCACAGCTTCTATGTTCCTGTTGTGAAATTCCATTTTGAACCTTCTCCCATGCAGGTTGGCATCTCACCTTTTCTTCGTTTGGTTCATAACTGAAAGAAAAACTAATTCTTATTTTATCATCACTTCCAAGGTCTTGG
OsPDIL5-4 ...................................................................----------------------------------------------------------------------------.......
OsPDIL5-4a ...................................................................----------------------------------------------------------------------------.......
6160 6170 6180 6190 6200 6210 6220 6230 6240 6250 6260 6270 6280 6290 6300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TAACTGAACTTCCAAAATCCTTCTCCCATTTCATCACAAATGTTTGTGCTATCATTGGAGGAGTTTTCACGGTGAGAGCACACACCCTTTTTTTATCTTTTCCTTTTCTCTGAATCCATCAAGTGTACATGGCTAATGAAATGACATGCG
OsPDIL5-4 .........................................................................-----------------------------------------------------------------------------
OsPDIL5-4a .........................................................................-----------------------------------------------------------------------------
6310 6320 6330 6340 6350 6360 6370 6380 6390 6400 6410 6420 6430 6440 6450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AATTCTAGGTTGCTGGAATACTGGATTCCATCTTCCACAACACCTTGCGGCTGGTTAAGAAGGTCGAGCTTGGGAAGAACATTTGAACAACAAGATTATTCTTCAGAAGCAGCCTCAAGCTTAGTGGTTGCACTAGTTTTAGTCATTATT
OsPDIL5-4 ----------............................................................................----------------------------------------------------------------
OsPDIL5-4a ----------............................................................................----------------------------------------------------------------
6460 6470 6480 6490 6500 6510 6520 6530 6540 6550 6560 6570 6580 6590 6600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 TTGTTATACCAGGGTAGGAATTCTTAATATATCAAGATGTGAAAGAGGGTAGTGGGTTGGCAGAGGAGGAGAAGTTGGGGTCATGATATATAGTAATAGTAGGTAGGGACCTCACTGTTTTGCTAAACGACTTCGTAAGAACAATAAGAC
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
6610 6620 6630 6640 6650 6660 6670 6680 6690 6700 6710 6720 6730 6740 6750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os07g34030 AGTGGATCCTTGTTAACTATACCCTACGTTAATTCGTCCAGACTTGTGTTGTGTTACTCTGTTTACTGTTGTGATGGCACCTGTCATTACTCGTTACTCTGGATTTGTCTTCCGTCTCAAAATTCATCTACGGTAACCAGTATTTTTAAG
OsPDIL5-4 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL5-4a ------------------------------------------------------------------------------------------------------------------------------------------------------
Intron X
Intron VIII
Exon XI
Exon IX
Intron XI
Intron IX
Exon XIIExon X
Intron XII
Intron X
Exon XIII
Exon XI
Intron XIII
Intron XI
Exon XIV
Exon XII
311
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 GGTGAATCCGACGTGGAGGCTCTCCATTGGTCCGCACACGCGTCCGTCCGTCTTCCTCGCGAGCGAACTTAAAATCGCAGTCGCACCCCGGCGCCGATCGCAAGGTCCACCTCCTCCCCTCTCCACTCCTTCTAGAACCTTCTCGACCCC
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 GCCATGCGCCCCGCGGTCGCCGCCGCCCTCCTCCTCGTCGCCGCCGCCGTCGCGGCCTCGCCGGTTTCCGCCCTCTACTCCGCCGGCTCTCCCGTCCTCCAGTTCAACCCCAACAACTTCAAATCCAAGGTCTCTCGATCGACTCCCCCC
OsPDIL2-3 ---..............................................................................................................................---------------------
OsPDIL2-3a ---..............................................................................................................................---------------------
OsPDIL2-3b ---..............................................................................................................................---------------------
310 320 330 340 350 360 370 380 390 400 410 420 430 440 450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TCACACTCTTCCCTTCCGTCTCCGGATCGGCTTCCCTTACTTGGGGGCTCTCGCTGAGTTCGAATTTCGGTTGGGGGTGCAGGTGCTGAACTCGAATGGGGTGGTGCTGGTGGAGTTCTTCGCGCCGTGGTGCGGGCACTGCCAGCAGCT
OsPDIL2-3 ----------------------------------------------------------------------------------....................................................................
OsPDIL2-3a ----------------------------------------------------------------------------------....................................................................
OsPDIL2-3b ----------------------------------------------------------------------------------....................................................................
460 470 480 490 500 510 520 530 540 550 560 570 580 590 600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 GACGCCGATTTGGGAGAAGGCCGCGGGCGTCCTCAAGGGCGTCGCCACGGTCGCCGCGCTCGACGCCGACGCGCACAAGGAGCTCGCGCAGGTGAGCAAACGTCCTGTTTGGCATCTCGGTTCATTGGGATTGGCTGATCTACCTTTTCC
OsPDIL2-3 ...........................................................................................-----------------------------------------------------------
OsPDIL2-3a ...........................................................................................-----------------------------------------------------------
OsPDIL2-3b ...........................................................................................-----------------------------------------------------------
610 620 630 640 650 660 670 680 690 700 710 720 730 740 750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TGGTGTGATCTGATCTCTGCATCAGTTGTTGCGTCAACGTAATTTGATCGAGCTAGGACATAACTGCGTATATGCAATGTGCAAACTCGACTCCATAAAAAAAAAAGTTACACCTGTTATGGGTTTTTTGTGTAGCTCTCAGTATCAGAG
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
760 770 780 790 800 810 820 830 840 850 860 870 880 890 900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 AGTACAATGCCACATTTGTTTGGTGGAGGAAATATGGGAAGATCAATGTTTGGTGTCTACACGTACTGATCAAAAAGGGGCGTCGTTTTTATTTTTTAAAGGAGGGCATTAACATCGGATATGAGTTATACAATACCGAATTAATGCATA
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
910 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040 1050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TTAAAATGCTGTAGCTGGGATTAATATTGATGGAATGTGGTGCATTGTTGATTTCATACAGTAAATTTATTTATTTTTTCACTTTTTCAGCATGGTGATTGGATGCTGTTCTCTTTTTTTTAAAAAAAATAACCTGGATTTTGAATTATA
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TTGAACGAATTAAGCATTTTAATTGGTGTTAATTAGGGTTTTATCTGTTTTTCTTTTGATCCGAAGGTGAGAGCGGCCTTGGTTAGTTTATTGCCAATAAAAGTGTATATGTGACTTTAGATGTGGGAAATGCATACTTTTATGAAGGAC
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
Exon I
Exon I
Exon I
Intron IIntron IIntron I
Exon II
Exon II
Exon II
Intron IIIntron IIIntron II
M
312
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 GAAACCGTGGAAGCCAATGGGTTGTGCAACTTTACAATATCTGATGCAACATTGTTTATTGGTGAATCAGAAAATTAGGTTATGTATCCCAGATTCTCTAACTGAAAATTTAGTCACTCATCAAATGAAATAAAGGCATCTATCCTGAGA
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 GAAGGCAAGCAAGAGTGAAAGATAGTTATGTACCTTTGCCATTGTACCTGCAGTGCGGTGATTTTGAGTCTCTGTCATATTTAGACATATATTTTTTTTTACAGTAATTTTACAGTATCATGCTTATTGACCAAGAGGCATTTTTTAGCT
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 ACGTTATATCTTTGTTTTGGTGAGTATTAGTCTTCTGTCTAGGCTAAATCAATAAAAACTGCACATTAGAACGTCTTCCACCATTGCCATTTGTAAATATTTTGACTGGATGCTATCAAGTTTTTCCTGATGCATGATGCCTTTGTTCTG
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770 1780 1790 1800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 CAGGAATATGGAATAAGAGGATTTCCAACTATTAAGGTGTTTGTTCCTGGAAAGCCTCCAGTAGATTATCAAGGAGCAAGAGATGTAAAGCCAATTGTTGAATTTGCTCTTTCACAGGTTTGTTGCAGCTTGCCCTATTTGATATATATA
OsPDIL2-3 ---..................................................................................................................---------------------------------
OsPDIL2-3a ---..................................................................................................................---------------------------------
OsPDIL2-3b ---..................................................................................................................---------------------------------
1810 1820 1830 1840 1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 AAAATGATATGTTGTTTTTACTTTTATTGCTCCTGTTCTATCTGCCACTCCACTAGTGTATACAACTACAAATCCTTTTCTGAAGATGTTAAGTGCTTCCTGGATTTTCACAGACCAAACTTATTCAGATTGCAATTGTCAGCTGAATGT
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
1960 1970 1980 1990 2000 2010 2020 2030 2040 2050 2060 2070 2080 2090 2100
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TGACATAAATAACTTTTATCTTAACATGAAGTATTACTAGGGGGGAAAAACAACAGATATCATATATCAACTGATACTAATGTCCTAGGTAGCTAATTGATTTTGCTATATATCTCCCTCAAATGCTAAACCTTTCAAACTTATCAACTT
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
2110 2120 2130 2140 2150 2160 2170 2180 2190 2200 2210 2220 2230 2240 2250
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TTTATATGGGATACTGGATATGAGGTGATGCAGTGATGCTTCTCAGTTCTTACCATGGTGGAGCTTTATGTACCTGCTCTTGCACAGCTGGTGCTTCTGGTAGCACCCATGATTTTCGTTAAAAAATACACTGTCTGTCGCTGCTGTGTT
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
2260 2270 2280 2290 2300 2310 2320 2330 2340 2350 2360 2370 2380 2390 2400
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 ACAGTGGGTTGCCATTTTACCTTTTTTTTTTGGGTATTTCTCTGTAAAAAATTTCAGCCTTGTGACACACGATATGAGTTTCCACTTTTAAATACAATGAATCTGTATCCTACAGGTCAAGGCTCTTCTTAGAGATCGGTTGAATGGAAA
OsPDIL2-3 -------------------------------------------------------------------------------------------------------------------...................................
OsPDIL2-3a -------------------------------------------------------------------------------------------------------------------...................................
OsPDIL2-3b -------------------------------------------------------------------------------------------------------------------...................................
Exon IIIExon IIIExon III
Intron III
Intron III
Intron III
Exon VIExon VI
Exon VI
313
2410 2420 2430 2440 2450 2460 2470 2480 2490 2500 2510 2520 2530 2540 2550
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 GACATCAGCAGGTTCAGGTGGCAAGAAATCAGGTGGTTCAAGTGAGAAAACTGAACCAAGTGCGTCAATAGAATTAAATTCTCAAAACTTCGATAAACTTGTCACCAAAAGCAAGGATCTTTGGATTGTTGAGTTCTTTGCACCATGGTA
OsPDIL2-3 ...................................................................................................................................................---
OsPDIL2-3a ...................................................................................................................................................---
OsPDIL2-3b ...................................................................................................................................................---
2560 2570 2580 2590 2600 2610 2620 2630 2640 2650 2660 2670 2680 2690 2700
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TGTGGCTTAATGCATTTATGGTTTTCATGTTGACTGATTTATTCTTTACTGTTGTTTCTGAACTTAACTCTGAATCCAAAAGTAGAGAAGGAAAGTGAAGAAAAACACAAAATTTCATACAATTTAGTCACATGTTAGTATTGTAGTGTG
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820 2830 2840 2850
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TACATTTTAGATCATTGCCCTTAAGATTATGTTGTTCAAGATAATAAGTAAATCCTGTTAACTGAAGACATTATTTGACTCATTAACTGTACTGGTCTTTTGTAAGGTGTGGGCACTGCAAGAAATTGGCACCTGAATGGAAAAAGGCTG
OsPDIL2-3 ----------------------------------------------------------------------------------------------------------............................................
OsPDIL2-3a ----------------------------------------------------------------------------------------------------------............................................
OsPDIL2-3b ----------------------------------------------------------------------------------------------------------............................................
2860 2870 2880 2890 2900 2910 2920 2930 2940 2950 2960 2970 2980 2990 3000
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 CAAAGAACTTGAAGGGCCAAGTGAAGCTAGGTCATGTGGATTGTGATGCTGAAAAGGTTCTTGCCCTTTTTCTATTTGGTCATAAGTTTTTGTTGTATGTGCATTTCTTGTTTGCAAATAATTTATACAAAATTATAAACTGAAATTTGA
OsPDIL2-3 ........................................................----------------------------------------------------------------------------------------------
OsPDIL2-3a ........................................................----------------------------------------------------------------------------------------------
OsPDIL2-3b ........................................................----------------------------------------------------------------------------------------------
3010 3020 3030 3040 3050 3060 3070 3080 3090 3100 3110 3120 3130 3140 3150
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 CAATGCATTGGATATTTCATCAATTCAAAGGACTTTGATAAAGGTATTGTAAAATAAAATTAAACTGAGGGTTAGCACTCTAGTTTATAGGGACACAATCAGGATATGCATAGGAAAGTACAAAATGCTGTATCAAGGAAAGCAATGGCC
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
3160 3170 3180 3190 3200 3210 3220 3230 3240 3250 3260 3270 3280 3290 3300
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TAACTTTATCCTAAGGCATTTGTTGGGATCTTATTTTGTGCTGTTTTAGGTTGTCTCTTTAACAGTTTAACATGATGTACTATGATTGAAGTTTTATGTTTTTTTTTCTAGAATACTAATTCAAGTGTTTGGTCAGTTGATAAAAGTTAC
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 CGAGTGAGTTTAAATGAACTTAGGCAGAAAATTCAGGCAGTGTATCAGTATATTTATAAAACACAGTTAATGTTTGACCTTTTTAAAGCATTCATGTGCGTTCTGTACAACATTTGTCAAACTATACAACCTAGTTTTCAAGTACCACTT
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
3460 3470 3480 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 ATGTAACATAACGTGTGACTTGAATTTTGCAGTCTTTGATGAGCAAGTACAAGGTAGAAGGCTTTCCTACTATTTTGGTATTTGGTGCCGATAAGGAGAGCCCATTCCCTTACCAGGGGGCTAGAGTTGCCTCTGCTATCGAGTCCTTTG
OsPDIL2-3 --------------------------------......................................................................................................................
OsPDIL2-3a --------------------------------......................................................................................................................
OsPDIL2-3b --------------------------------......................................................................................................................
Intron IVIntron IV
Intron IV
Exon V
Exon V
Exon V
Intron V
Intron V
Intron V
Exon VIExon VI
Exon VI
314
3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 3730 3740 3750
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 CATTGGAGCAGTTGGAAGCCAATGCTGCTCCACCTGAAGTTTCTGAGTTGACTGGCCCAGTAAGTTTTCGTGATATTAACAAAGTTATCCTTTTGTGGAGACAATGGTCTGGGATTGATGTTTTGACGAGGTGGTTTGGCTTCTGTAGGA
OsPDIL2-3 ............................................................-----------------------------------------------------------------------------------------.
OsPDIL2-3a ......................................................................................................................................................
OsPDIL2-3b ............................................................-----------------------------------------------------------------------------------------.
3760 3770 3780 3790 3800 3810 3820 3830 3840 3850 3860 3870 3880 3890 3900
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 CGCCATGGAAGAGAAATGTGCTTCTGCTGCCATTTGCTTTGTATCTTTCCTTCCAGATATCTTGGATTCAAAGGCCGAAGGAAGAAACAAGTACCTTGAGCTGCTATTATCTGTTGCTGAGAAATTTAAAAAGAGTCCATACAGGCAAGA
OsPDIL2-3 ................................................................................................................................................------
OsPDIL2-3a ................................................................................................................................................------
OsPDIL2-3b ......................................................................................................................................................
3910 3920 3930 3940 3950 3960 3970 3980 3990 4000 4010 4020 4030 4040 4050
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TTCAACTAAACTTGTTGTGGACTTTATACCATTCTACCTATCATTCCTTGTCTTGTTATGTCTTTTGAGATAAAAAAATCCACAGTTAAATTGATACATTAATTTCCTTTTGCTATTGAATATTGATTGACAAAAAGATTGATAGTAACC
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b .........................................................................-----------------------------------------------------------------------------
4060 4070 4080 4090 4100 4110 4120 4130 4140 4150 4160 4170 4180 4190 4200
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TTCATATATTTGAAACACCTTAAAAGAATTAAATGACTTAGTTCGATAATGTTATATGCCAAGGCGTGTATACTATAAGCTCTAAACATAATGGAGATTTTATATATTATATATGCAAATCTGCCATGTGACTGAGTTCTATGTTGAGAA
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
4210 4220 4230 4240 4250 4260 4270 4280 4290 4300 4310 4320 4330 4340 4350
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 GTTTCCTCAGACAAATCCATTCACTTAGTTAATGCCTTGAAATCTCTTCCGTTTCTTGAGAAAGCCATTCTGAGCTAGAAAAAGCATTGTCTTGCAGTTTTGTCTGGACAGCTGCTGGGAAGCAAGCTGATCTTGAGAAGCAAGTTGGAG
OsPDIL2-3 -------------------------------------------------------------------------------------------------.....................................................
OsPDIL2-3a -------------------------------------------------------------------------------------------------.....................................................
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
4360 4370 4380 4390 4400 4410 4420 4430 4440 4450 4460 4470 4480 4490 4500
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TTGGTGGTTATGGCTATCCAGCGATGGTTGCTCTCAACGTGAAAAAAGGCGCATATGCTCCACTCCGTAGTGCTTTCCAGCTTGATGAAATAACGTAAGTTCCTTGTGCATAGAAAATTAATTGCTGCTTAAGGTTCATTTGCCAGATGT
OsPDIL2-3 ..............................................................................................--------------------------------------------------------
OsPDIL2-3a ..........................................------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
4510 4520 4530 4540 4550 4560 4570 4580 4590 4600 4610 4620 4630 4640 4650
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TTGATAACAAAACCGGTCTCCTGCAGTGAGTTTGTGAAGGAAGCAGGGCGCGGTGGGAAGGGTAATCTTCCTTTGGACGGTACCCCAACGATAGTCCAATCTGAACCATGGGACGGCAAAGATGGTGAGGTTATTGAAGAGGACGAATTC
OsPDIL2-3 --------------------------............................................................................................................................
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
4660 4670 4680 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TCCCTTGAGGAGCTGATGGCCGACAACTCTCCAGTAAATGACGAGTTGTGATTCTGATAATTCCGGAAATGATGTTAGAGTTGATTAGCCAGACTGTAAATCTGAGTAACACTAATGCTTCACTCGTGTCAACTGTACAATTTGGGCGGG
OsPDIL2-3 ...................................................---------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
Intron VI
Intron VI
Exon VII
Exon VII
Intron VIIIntron VI
Eonx VIIIEonx VII
Intron VIII
Exon IX
315
4810 4820 4830 4840 4850 4860 4870 4880 4890 4900 4910 4920 4930 4940 4950
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 ACAATTTTCAGAATGCTCCTTTGGTGTTATTACAGTACATTTTAGTTGAAGAAAAACATCGTTTGTGGTCTTTTAGGAGGGATGCTTTTCTCTGGGAAATAAAACTACTTCGTAATCTAGATATTTTGCCTCAACGTGTTTTCCTTCAAA
OsPDIL2-3 ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3a ------------------------------------------------------------------------------------------------------------------------------------------------------
OsPDIL2-3b ------------------------------------------------------------------------------------------------------------------------------------------------------
4960 4970 4980
. . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
Os09g27830 TAGAAATCTATGCTTTATGCTGTTGTCTGT
OsPDIL2-3 ------------------------------
OsPDIL2-3a ------------------------------
OsPDIL2-3b ------------------------------
Dots indicate the sequences of exons are identical to the corresponding part of genomic loci. Broken lines indicate the 5’ and 3’ extra regions compared with the CDSs, and introns. A: OsCYP24-1 and OsCYP24-2 generated from LOC_Os06g49480; B: OsCYP46 and OsCYP50 generated from LOC_Os07g37830; C: OsCYP40 and OsCYP28 generated from LOC_Os08g19610; D: OsCYP73 and OsCYP58 generated from LOC_Os08g44330; E: OsCYP18-2, OsCYP19-3, and OsCYP20 generated from LOC_Os08g44520; F: OsPDIL2-2 and OsPDIL2-2a generated from LOC_Os01g23740; G: OsPDIL1-3 and OsPDIL1-3a generated from LOC_Os02g34940; H: OsAPRL3 and OsAPRL3a generated from LOC_Os02g51850; I: OsPDIL5-1 and OsPDIL5-1a generated from LOC_Os03g17860; J:
OsPDIL2-1 and OsPDIL2-1a generated from LOC_Os05g06430; K: OsQSOXL1 and OsQSOXL1a generated from LOC_Os05g47930; L: OsPDIL5-4 and
OsPDIL5-4a generated from LOC_Os07g34030; M: OsPDIL2-3, OsPDIL2-3a, OsPDIL2-3b generated from LOC_Os09g27830.
316
Appendix VI Summary of intron sizes (bp) of Cyp genes in rice*
Intron
I Intron
II Intron
III Intron
IV Intron
V Intron
VI Intron
VII Intron
VIII Intron
IX Intron
X Intron
XI Intron
XII Intron
XIII Intron
XIV
OsCYP18-2 120 158 338 591 537
OsCYP19-3 120 158 302 591 537
OsCYP20 1352 95 338 591 537
OsCYP22-1 120 416 247 683 199 308
OsCYP22-2 202 195 93 501 114 131
OsCYP23 645 140 236 93 177 74
OsCYP24-1 249 396 93 466 108 99
OsCYP24-2 234 396 93 466 108 99
OsCYP25 106 101 589 543 627
OsCYP26-1 801 85 363 80 354 787
OsCYP26-2 116 490 816 1756 104 381
OsCYP27 99 752 104 578 87 86
OsCYP31 133
OsCYP40 2716
OsCYP43 1951 382 90 650 600 81 75
OsCYP44 1698 172 115 195 282 76 86
OsCYP46 99 88 145 118 102 1211 185 244 217 225 101
OsCYP47 1076 161 562 260 107 100
OsCYP49 291 973 317 453 632 423 131 70 483
OsCYP50 88 145 118 102 1211 185 244 172 225 101
OsCYP51 95 90 91 99 266 98
OsCYP57 263 109 141 82 1178 186 555 169 104
OsCYP58 94 454 97 81 317 136 138 244 91
OsCYP63 72 1235 864 122 103 131 305 92 253 82 1276 354 1467 76
OsCYP65 526 79 147 97 138 360 89 163 82 640 236 1215 72
OsCYP70 115 526 267 88 114 555 80 89 663 412 80 568
OsCYP73 86 323 162 94 454 97 81 317 136 138 244 91
The sizes of introns were calculated based on the gene structures (Fig 5.1) generated with gene structure display server program (http://gsds.cbi.pku.edu.cn).
317
Appendix VII Summary of intron sizes (bp) from PDILs genes in rice*
Intron
I Intron
II Intron
III Intron
IV Intron
V Intron
VI Intron
VII Intron
VIII Intron
IX Intron
X Inron
XI Intron
XII Intron
XIII Intron
XIV
OsPDIL1-1 639 104 103 141 290 82 110 190 112
OsPDIL1-2 94 107 130 84 260 89 115 119 101
OsPDIL1-3 113 523 508 219 134 426 429 327
OsPDIL1-3a 113 523 508 219 134 426 429 327
OsPDIL1-4 85 107 98 87 167 125 466 75 79 596 811
OsPDIL1-5 99 140 267 172 100 321 74 489 113 652 78
OsPDIL2-1 479 210 89 344 93 92 243 82 86 90
OsPDIL2-1a 479 210 89 344 93 92 243 82 83 90
OsPDIL2-2 1125 193 86 213 107 104 208 91 91 106
OsPDIL2-2a 1125 193 86 213 107 104 208 91
OsPDIL2-3 103 1112 598 259 576 89 400 82
OsPDIL2-3a 103 1112 598 259 576 400
OsPDIL2-3b 103 1112 598 259 576 89
OsPDIL5-1 564 98 917
OsPDIl5-1a 564 98
OsPDIL5-2 655 92 229 279
OsPDIL5-3 468 87 235 243
OsPDIL5-4 332 612 634 542 339 164 82 1288 125 207 85 121 76 87
OsPDIL5-4a 339 164 82 1288 125 207 85 121 76 87
OsQSOXL1 1345 107 316 175 109 88 74 441 116 81 145
OsQSOXL1a 1345 107 316 175 109 88 74 441 116 81
OsAPRL1 295 735
OsAPRL2 587 82 207
OsAPRL3 804 93 285
OsAPRL3a 804 93
OsAPRL4 933 83 363
OsAPRL5 1096 108 388
OsAPRL6 1285 85 333
The sizes of introns were calculated based on the gene structures (Fig 6.1) generated with gene structure display server program (http://gsds.cbi.pku.edu.cn).
318
Appendix VIII Screen captures showing putative wheat orthologues at PDILs and Cyps loci in rice
A
B
C
D
E
F
G
H
319
I
A: putative wheat orthologue (CSU046BE444859) at the OsPDIL2-2 locus in rice; B: putative wheat orthologue (UCD078BF201426) at OsPDIL5-2; C: putative wheat orthologue (UCD078BF201426) at OsPDIL5-3; D: putative wheat orthologue (NDS021BE500255) at the OsQSOXL1; E: putative wheat orthologue (KSU035BE403404) at OsAPRL1; F: putative wheat orthologue (NDS252BE445181) at OsAPRL6; G: Putative wheat orthologues (KSU027BE590822, NDS021BE406148) at locus (LOC_Os0649480) encoding OsCYP24-1 and OsCYP24-2 in rice; H: Putative wheat orthologue (UMW213BE497013) at locus (LOC_Os01g18210) encoding OsCYP25 in rice; I: Putative wheat orthologue (KSU004BE585547) at locus (LOC_Os02g10970) encoding OsCYP49 in rice. The captures were obtained from Rice Genome Browser in MSU Rice Genome Annotation (Osa1) Release 6 (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice ).
320
Appendix IX PDI gene family markers associated with quality QTLs in rice
PDI name Gene position in Gramene Map (bp) Colocalized QTLs Map set
Position (cM)
QTLs in Gramene Map (bp)
Flanking marker
Markers in QTL Maps
Markers in Gramene sequence
OsPDIL1-2 Chr4: 21515572-21512924 AROMA (CQAP2)
Cornell IR64/Azu DH QTL 2001 69.6 -135.4
21227137-29027171 RG788 69.6 N/A
OsPDIL5-2 Chr4:21277599-21275073 RM317 135.4 29027022-29027171
HULCL (AQGD023) NDSU EM93/ SS18 BC QTL 2004 16.8-160.7
18809314-35117807 RM167B 16.8 N/A
RM559 160.7 35117639-35117807
OsPDIL1-3 Chr2:20956031 - 20951715
CRKGRPCENT (AQEE018)
Cornell IR64/IRG105 QTL 2003 126.9-168.3
19865083-27610063 RG157 126.9 19865083-19865569
OsPDIL5-3 Ch2:20694520-20696830 RM221 168.3 27609877-27610063
HEADR (AQEE014) Cornell IR64/IRG105 QTL 2003 126.9-168.3
19865083-27610063 RG157 126.9 19865083-19865569
RM221 168.3 27609877-27610063
GRLGWDRO (AQGB055) JKU Aso/IR24 QTL 1996 55.9-77.9
18495134-22596902 XNpb67 55.9 N/A
XNpb132 77.9 N/A
SDLGWDRO (AQDH010)
IRRI IR64/Azu DH QTL 2003 40.6-63.5
19865083-24566168 RG157 40.6 19865083-19865569
RZ318 63.5 24565710-24566168
OsPDIL1-5 Chr6: 3198484 - 3202590**
GRLGWDRO (CQAL18)
CNHZAU Zh97/Ming63 RI QTL 2002 18.5-49
2260721-3536853 R1952 18.5 2260721-2260991
C226 49 3536009-3536558
ALKDIG (AQBP005) IGCN ZYQ18/JX17 DH QTL 1998 34.2-38.3
3168374-4160798 RM204 38.3 3168374-3168547
RM225 34.4 3416533-3416728
G165 36.3 4160454-4160798
AMYCN (AQFU016) LSU Caiapo/IRGC103544 QTL 2004 0-116
1764586-5425631 RM190 0 1764586-1764729
RM253 116 5425408-5425602
GELCON (AQGA022) CNHAU Zhen97/ H94 QTL 2005 0-39.6
441616-5425631 RM508 0 441616-441850
MRG5119 39.6 N/A
ALKDIG (AQFU018) LSU Caiapo/IRGC103544 QTL 2004 0-116
1764586-5425631 RM190 0 1764586-1764729
RM253 116 5425408-5425602
321
HEADR LSU Caiapo/IRGC103544 QTL 2004 0-116
1764586-5425631 RM190 0 1764586-1764729
RM253 116 5425408-5425602
ENDFLR (AQCV038) JRGP Nip/Kas F2 QTL 2000 10.7-13.5
2456191-3864403 V30B 10.7 N/A
G8021 13.5 N/A
ENDFLR (CQAL10) Zh97/Ming63 CNHZAU F2 QTL 1999 33-39.3
2260721-3536853 R1952 33 2260721-2260991
C226B (C226) 39.3 3536009-3536558
SDLG (AQT015) JHPRC Rei/Yamd QTL 2002 0-20.9
3168374-6230185 RM204 0 3168374-3168547
RM276 29 6230045-6230185
PROTCN (AQFU022) LSU Caiapo/IRGC103544 QTL 2004 0-116
1764586-5425631 RM190 0 1764586-1764729
RM253 116 5425408-5425602
OsPDIL2-1 Chr5: 3276014-3273106 GRWD (AQGB029) JKU Aso/IR24 QTL 1996 8.4-31.4
1839408-6679640 R3166 8.4 1839408-1839741
R569 31.4 6679190-6679332
GRLGWDRO (AQGB043) JKU Aso/IR24 QTL 1996 8.4-31.4
1839408-6679640 R3166 8.4 1839408-1839741
R569 31.4 6679190-6679332
OsPDIL2-2 Chr1:13336222-13339661 HEADR (AQFU004)
LSU Caiapo/IRGC103544 QTL 2004 16.2-24.3
12284223-24299406 RM140 16.2 12284223-12284522
RM5 24.3 24299213-24299406
SDLGWDRO (AQFU026)
LSU Caiapo/IRGC103544 QTL 2004 24.3-31
24299213-27663181 RM5 24.3 24299213-24299406
RM246 31 27663068-27663166
ENDFLR (AQCV031) JRGP Nip/Kas F2 QTL 2000 38.8-52.1
6734684-26863059 Y3853L 38.8 N/A
C1211 52.1 N/A
OsPDIL5-1 Chr3:9934130-9932108 ENDFLR (AQCV036)
JRGP Nip/Kas F2 QTL 2000 40.3-46.6
8389519-10303012 C63 40.3 8390337-8390403
C2045 46.6 10301490-10301584
GRLGWDRO (AQFA014)
Cornell V20A/glab QTL 2004 31.2-76.9
9825151-23044841 RZ16A 31.2 9825151-9825191
RZ598 76.9 22786808-22786864l
GRLG Cornell V20A/glab QTL 2004 31.2-76.9
9825151-23044841 RZ16A 31.2 9825151-9825191
RZ598 76.9 22786808-22786864l
OsPDIL5-4 Chr7:20339546- PERICARPCL NDSU EM93/SS18 BC 0-120.5 412967- RM295A 0 412967-413153
322
20345697 (AQGD030) QTL 2004 25651967
OsAPRL1 Ch7:19411494-19408595 RM18 120.5 25651810-25651967
PERICARPCL (AQGD028)
NDSU EM93/SS18 BC QTL 2004 0-146.4
412967-29560750 RM295A 0 412967-413153
RM172 146.4 29560592-29560750
CLGRPCENT (AQEE005)
Cornell IR64/IRG105 QTL 2003 57.4-94.5
6778415-21870655 RG477 57.4 6778514-6779120
RM336 94.5 21870504-21870655
OsQSOXL1 Chr5:27395700-27391291 MR (CQR3)
CNHZAU Zh97/Ming63 RI QTL 2002 114.4-127.6
27321395-28686006 C246 114.4 N/A
C1447 127.6 28684502-28684690
CULMSTR (AQFP018)
CAU IRAT109/YUEFU QTL 2004 73.2-77.8
7741675-27955848 R521 73.2 27955480-27055848
R566 77.8 7741675-7741816
OsAPRL2 Chr6: 6221709-6223415
SDLGWDRO (AQFU027)
LSU Caiapo/IRGC103544 QTL 2004 116-204
5425408-24035615 RM253 116 5425408-5425602
RM162 204 24035491-24035615
ALKDIG (AQFU019) LSU Caiapo/IRGC103544 QTL 2004 116-204
5425408-24035615 RM253 116 5425408-5425602
RM162 204 24035491-24035615
GELCON (AQEE023) Cornell IR64/IRG105 QTL 2003 62.2-85.1
5425408-6399814 RM253 62.2 5425408-5425602
RM402 85.1 6399680-6399813
CONVIS (CQAG19)** IGCAS ZYQ8/JX17 DH QTL 2000 32.8-50.1
5425408-9284025 CT506 32.8
C235 5425408-5425602
C235 50.1 9282505-9282553
CPVIS (CQAG9) IGCAS ZYQ8/JX17 DH QTL 2000 32.8-50.1
5425408-9284025 CT506 32.8
C235 5425408-5425602
C235 50.1 9282505-9282553
HPVIS (CQAG5) IGCAS ZYQ8/JX17 DH QTL 2000 32.8-50.1
5425408-9284025 CT506 32.8
C235 50.1 9282505-9282553
SDLG (AQT015) JHPRC Rei/Yamd QTL 2002 0-20.9
3168374-6230185 RM204 0 3168374-3168547
RM276 29 6230045-6230185
OsAPRL3 Chr2:31755530-31753413 GRWD (AQCV011)
JRGP Nip/Kas F2 QTL 2000 128.3-139.3
30068383-32036692 C601 128.3 N/A
323
C560 139.3 N/A
CRUSHGRPCENT (AQEE020)
Cornell IR64/IRG105 QTL 2003 154.4-182.9
25865334-32774553 RM362 154.4 25865334-25865568
RM250 182.9 32774365-32774538
GRLG (AQCV001) JRGP Nip/Kas F2 QTL 2000 128.3-139.3
30068383-32036692 B252 128.3 N/A
C560 139.2 N/A
OsAPRL4 Chr8:19597590-19599760 ENDFLR (AQGB080) JKU Aso/IR24 QTL 1996 6.5-30.4
18864478-26225545 G1149 6.5 26224841-26225086
R1394 30.4 18864478-18864562
AROMA (CQAP1) Cornell IR64/Azu DH QTL 2001 24.8-114.9
2109496-24661864 RG20 24.8
RM38 2109496-2109612
RZ66 114.9
RM308 24661732-24661864
BRWRPROT (AQFA019)
Cornell V20A/glab QTL 2004 41.5-51.2
16241105-19964566 RG1034 41.5 16241105-16241545
RG28A 51.2 N/A
OsAPRL6 Chr12:21635926 - 21633321 ENDFLR(AQCV035)
JRGP Nip/Kas F2 QTL 2000 64.7-89.5
18867450-23318207 C50732S 64.4 19000811-19001119
R10289S 86.5 23300722-23301169
AROMA (CQAP3) Cornell IR64/Azu DH QTL 2001 49.5-102.8
8826555-26108004 RM101 49.5 8826555-8826854
RM235 102.8 26107904-26108004
GELTZTMP (AQGA042)
CNHAU Zhen97/ H94 QTL 2005 89.4-92.9
21454591-21742415 RM309 89.4 21454591-21454758
RMG2326 92.9
RM3326 21742024-21742415