arabidopsis molecular genetics hort 301 – plant physiology october 31, 2007 reading 1 (alonso and...
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Arabidopsis Molecular Genetics HORT 301 – Plant Physiology
October 31, 2007Reading 1 (Alonso and Ecker (2006) Nature Reviews Genetics
Reading 2 (Page and Grossniklaus (2002) Nature Reviews Genetics
Function of all plant genes
Assumption – majority of genes in plants have conserved function, including those in crops
Lecture outline:
Genetic terminology
Arabidopsis thaliana, the current plant genetic model system
Resources for gene function determination
Phenotypic selection or screening of a T-DNA mutant population – forward genetics, identification of the mutated gene
Candidate gene based on predictions of function (phenotype) – reverse genetics
Terminology
Genetics – study of heredity/inheritance and function of genetic material
Gene – individual functional unit of the DNA that includes the promoter and encodes the messenger mRNA that is translated into a protein, i.e. locus
Locus – usually two alleles at each, contributed by two homologous chromosomes in a diploid (2n) organism
Molecular genetics – application of molecular biology to genetics, i.e. gene identification by DNA structure, genetic engineering
Genotype (genes) to phenotype (appearance and function)
Inheritance – single gene dominant and recessive phenotypes
Griffiths AF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM. 1996. An Introduction to Genetic Analysis. 6th ed. W.H. Freeman & Company
Yellow – 416 (3)Green – 130 (1)
Y (dominant) and y (recessive) are alleles
Allele – alternative state of a gene that may be different in each of the two chromosomes of the pair, dominant (Y) or recessive (y) allele
Mutation – change in DNA structure of a gene, alteration in gene function
WT siz1-2 siz1-3
Plant breeders improve crops by transferring “mutations” that confer better phenotypes/traits!!!!
See the maize example
Arabidopsis thaliana (L.) Heynh, plant genetic model – (Brassicaceae, mustard or crucifer family) common name Arabidopsis, rosette-type plant
25.1 (A) The shoot apical meristem in Arabidopsis thaliana; (B) An Arabidopsis plant
Morphological, developmental and molecular genetic attributes of Arabidopsis that make this plant a genetic model:
Small size – numerous plants can be grown in a small area
Short life cycle – six to eight weeks, 6 to 8 generations per year
Produces numerous seed – several thousand seeds per plant
Diploid (2n) – n = 5 (chromosomes)
Self-fertile – amenable to classic genetic manipulation by self or cross pollination
Natural variation – better alleles for certain traits in other accessions or ecotypes
Transformable using Agrobacterium tumefaciens – foreign DNA is easily transferred into the genome (complete set of genes and accompanying DNA)
Genome size is relatively small - ~120 x 106 bp, 26,819 genes encode proteins, 31,762 total including genes that encode miRNA, pseudogenes, and transposable elements, etc. http://www.arabidopsis.org/portals/genAnnotation/genome_snapshot.jsp
Genome sequence information is available, also cDNA sequence, http://signal.salk.edu/cgi-bin/tdnaexpress
Bioinformatic information, http://signal.salk.edu/, http://mips.gsf.de/proj/plant/jsf/index.jsp
mRNA expression data, https://www.genevestigator.ethz.ch/at/
Mutations for nearly every gene – mutants are available, http://www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm
Genetic resources for gene function identification - T-DNA insertional tagging mutagenesis - Agrobacterium-mediated transformation
Agrobacterium tumefaciens – crown gall disease, bacterial pathogen of plants that transfers DNA (T-DNA) into the plant genome during the infection process
Tumor cells produce carbon and nitrogen sources for use by the bacteria
Agrobacterium infection and tumor development
“Disarmed” Agrobacterium strains are used for genetic manipulation (engineering) of plants – bacteria are no longer pathogenic but are still capable of T-DNA transfer
T-DNA is inherited as a single dominant gene (locus)
T-DNA binary vector (plasmid) composition, e.g., pSKI015 - right border (RB) and left border (LB)
DNA between the borders is inserted into the plant genome (DNA), selectable marker gene
Selectable marker gene – e.g. herbicide resistance gene to “select” transformed plants
Insertions are random, 1.5 insertions per event
4X 35ST-DNA Vector – *pSKI015
Transformation
Mutant Plants
Plant Genomic DNA T - DNA
or
Disruption
Activation
LB 3’-ocs-bar-mas-5’ OriC RB
Agrobacterium T-DNA insertional (tagging) mutagenesis
Activation sequence – e.g., 4X 35S that can activate expression of a native gene depending on the insertion position
Primary interest is to alter the function (cause a mutation) of every gene by T-DNA insertional mutatgenesis
It is estimated that ~300,000 random insertions will “saturate the genome”, a mutation in each gene, a population of 300,000 plants
Process is referred to as “tagging”
“Tag” – insertion of the T-DNA “tags” the region in the genome because the T-DNA sequence can be located in the genome because of sequence
Identification of the flanking sequence
Floral transformation of Arabidopsis
Each seed that is transformed has a unique mutation
Transformation procedure - inflorescences are dipped into a solution containing Agrobacterium
Plants are grown in the greenhouse and seeds are collected
Selection of transformed plants – typically based on resistance to a toxic agent (transgene expression results in detoxification), in this instance the herbicide bialaphos
Population is maintained through seed, i.e. inherited
Generation of T-DNA tagged population
Herbicide selection of transformantsPropagation and collection of
seed
Phenotypic screening of the T-DNA mutant population – forward genetics
Identify Salt Responsive Mutants in a T-DNA Insertion Population
T1 GenerationSelect Transformed Plants (Heterozygous, BialaphosR, ~0.2%)
T2 Generation
Primary Screen for Mutants (NaCl sensitivity or insensitivity)
T3 GenerationConfirm Genetic Stability of Phenotype (Homozygous,
Recessive/Dominant)
Locate the “Tag” and Identify the Flanking Genomic Sequence
Root Development(C24)
Luciferase Imaging(C24RD29A::LUC)
Shoot Development(Col-0 sos3-1)
Stress StressStress
Isolation of NaCl mutants using different approaches
Identification of the mutation in Arabidopsis genome – location of the T-DNA insertion (tag) and determination of flanking sequence
Most mutations are recessive, both alleles of the locus are homozygous for the mutation
T -DNAL B R B
L B primer
random primer
random primer
P roc edure for loc ating T -DNA in the Arabidops is g enome
---AATAC AG T G C C G TG AC T T T G T T C T TAAC TC T G G G G C AT T T AT T C C AC TG T T G C ATC AG C TG A------
e.g. F lanking sequence : –importin protein (At5g49310)
R B primer
P C R product
S equence of T -DNA S equence of unknown gene
(3) B las t-s earc h of identified flanking s equenc e in Arabidops is g enome databas e (http://www.nc bi.nlm.nih.g ov/B L AS T /)
(1) T AIL -P C R
(2) S equenc ing
T -DNAL B R B
?
-importin
-importin gene – At5g49310
Nature (2000) 408:796-815
Arabidopsischromosomes
Reverse genetics – candidate gene identification based on prediction of function and phenotypic effects
Ca2+/CaM regulated transcription factor that regulates drought stress responses of plants, GTL family
R evers e genetics
(1) S elect a gene or genes (ex. Arabidopsis G T -element binding transcription factor family)
At1g76880 (GTL6)
At1g33240 (GTL1)
At1g76890 (GT-2)
At5g28300 (GTL2)
At5g03680 (GTL3/PTL)
At3g10000 (GTL4)
At5g47660 (GTL5)
At2g33550
At1g31310
At3g25990
At1g13450 (GT-1)
At5g01380 (GT-3a)
At2g38250 (GT-3b)
At5g63430
At3g10040
At1g76870
At1g2120099
100
100
100
100
66
56
54
20
75
79
54
46
97
G T -2 /G TL
G T -1
Phylogenetic dendogram
Mutant lines are available to the public
(2) Generate a collection of mutations in selected genes: Arabidopsis Biological Resource Center (ABRC) provides T- DNA insertion
lines in Arabidopsis genome to public ( www.arabidopsis.org )
Gene T-DNA mutations Insertion position
AtGT-2
(At1g76890)
gt2-1 (salk_014451) Exon
gt2-2 (salk_035328) 3’ UTR
AtGTL1
(At1g33240)
gtl1-1 (salk_005972) Exon
gtl1-2 (salk_044308) Intron
gtl1-3 (salk_101901) 5’ UTR
AtGTL2
(At5g28300)
gtl2-1 (salk_087253) Promoter
gtl2-2 (salk_020059) 3’ UTR
AtGTL3
(At5g03680)
gtl3-1 (salk_144638) Intron
gtl3-2 (salk_010031) Promoter
AtGTL4
(At3g10000)
gtl4-1 (salk_058993) Exon
gtl4-2 (salk_145331) Intron
AtGTL-5
(At5g47660)
gtl5-1 (salk_049268) Promoter
gtl5-2 (salk_078330) Intron
AtGTL-6
At1g76880
gtl6-1 (salk_106258) Exon
gtl6-2 (salk_072465) Exon
AtGTL1 (At1g33240)gtl1-2
L RLLB
L+LB L+LB L+LBL+ LB L+ LB L+ LB
no T-DNA
homozygous heterozygous
No T-DNA homozygous heterozygous
Select homozygous T - DNA insertion line
Evaluate phenotypes
(3) Tes t phenotypes of mutations (F or example : Drought s tres s)Col-0 (wild type) gtl1-2 gtl1-3
0
20
40
60
80
100
Col-0 gtl1-2 gtl1-3
Sur
viva
l (%
)
R evers e genetic approach for G T L 1 gene revealed that the function of G TL 1 is important for drought adaptation in Arabidops is