plant molecular genetics and genetic transformation hort 301 – plant physiology november 1, 2010...
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Plant Molecular Genetics and Genetic TransformationHORT 301 – Plant Physiology
November 1, 2010Taiz and Zeiger – Chapter 2, Smith et al. (2010) – Chapter 9
Function of all plant genes – omics; genome, transcriptome, proteome
Allelic variation – Le, GA20 →GA1
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Mutations - phenotype
Molecular genetics Genetics – study of heredity/inheritance and function of genetic material Molecular genetics – gene identification, structure and function, biotechnology Genotype – genetic constitution, genesPhenotype – appearance and other function(s) resulting from gene functionGene – functional DNA unit, promoter and coding sequence, i.e. locus
Griffiths AF, et al. 1996. An Introduction to Genetic Analysis. 6th ed.
Locus – usually two alleles at each, contributed by two homologous chromosomes in a diploid (2n) organism, dominant (Y) and recessive (y) alleles
yy
yy
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Mutation – change in DNA structure of a gene
WT siz1-2 siz1-3
Mutation may result in loss (reduction in)- or gain-of-function, or may not have an effectFunction is inferred based on deviation from normal
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Smith et al. (2010) Plant Biology
Fruit size regulated by reduced function of a cell division inhibitorPlant breeders improve crops by transferring “mutations” that confer better phenotypes/traits!
Domestication often selects against fitness
Gene function based on gain- or loss-of (reduced)-function by transgene expression
Dwarfism caused by reduced GA levels, over-expression of GA oxidase
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Vitamin A production by transgene overexpression – trans-kingdom gene transfer
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Smith et al. (2010) Plant Biology
Gene (RNA) silencing – natural viral RNA suppression, plant defense
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Transgene-induced gene silencing – GFP
Smith et al. (2010)
Determining gene function based on mutagenesis – transgene tagging
Forward genetics – phenotype is selected and then the mutation in a specific locus is identified
WT siz1-2 siz1-3
Reverse genetics – mutation in a specific locus is identified and then the phenotype is determined
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Genetic transformation of plants – functional analysis by gene transfer and inheritance of transgene
Smith et al. (2010) Plant Biology
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Agrobacterium causes crown gall
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Agrobacterium T-DNA is integrated into the plant genomeTumorization - auxin and cytokinin biosynthesisTumor growthOpine biosynthesis
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Agrobacterium infection and integration processes
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Smith et al. (2010) Plant Biology
“Disarmed” T-DNA vector for plant genetic transformation – elimination of genes that cause tumor formation and growth, opine biosynthesis Binary system includes a vir gene plasmid for replication, infection and integration
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DNA delivery “vehicles” for transformation of plant cells
Plant Vector w/Multiple Expression Cassettes Reporter Marker
Target
Plant Expression Cassette
Enhancer/Promoter
Coding sequence
Terminator/ AAA/3’-UTR
Leader5’-UTR
T-DNA is integrated randomly in the plant nuclear genome and is inherited as a single dominant gene (locus)
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Plant regeneration by organogenesis – regenerate plants from individual cells (independent transformation events
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Transformation of sorghum cells and plant regeneration via somatic embryogenesis
CONTROL TRANSFORMED
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4X 35ST-DNA Vector – *pSKI015
Transformation
Mutant Plants
Plant Genomic DNA T - DNA
or
Disruption
Activation
LB 3’-ocs-bar-mas-5’ OriC RB
Arabidopsis mutations by T-DNA “tagging” - pSKI015 insertion results in activation or gene disruption
17T-DNA is inserted randomly into the genome
Floral transformation of Arabidopsis
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Generation of T-DNA tagged population of plants
Herbicide selection of transformantsPropagation and collection of seed
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Root Development(C24)
Luciferase Imaging(C24RD29A::LUC)
Shoot Development(Col-0 sos3-1)
Stress StressStress
Isolation of NaCl mutants using different approaches
Forward genetic approach – phenotypic selection or screening of the T-DNA mutant population
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Identification of the mutated gene – locate the T-DNA insertion (tag) and determine flanking sequence
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 TC T TAAC TC TG G G G C AT T TATT C C AC TG T TG 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) TAIL -P C R
(2) S equenc ing
T -DNAL B R B
?
-importin
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-importin gene – At5g49310
Nature (2000) 408:796-815
Arabidopsischromosomes
Molecular breeding is a forward genetic approach! 22
Reverse genetic approach – determine function of a candidate gene
GTL family of putative Ca2+/CaM regulated transcription factors involved in stress responses?
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
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Mutant lines are available to the publicArabidopsis Biological Resource Center (ABRC, www.arabidopsis.org) T-DNA insertion lines
Genotyping
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+R L+R L+RL+ LB L+ LB L+ LB
no T-DNA-DNA
homozygous heterozygouss
No T-DNA homozygous heterozygous
Select homozygous T- DNA insertion line
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Evaluate phenotype
(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
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Biotechnology
Majority of GM crops (corn, cotton, soybeans) are insect or herbicide resistant
Virus resistance
Pharmaceuticals and nutriceuticals (biofortification)
Stress tolerance
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Millions of Hectares of GM Crops 2008 – Total 124 million ha (315A)
• USA 62.5• Argentina 21.0• Brazil 15.8• Canada 7.6• India 7.6• China 3.8• Paraguay 2.7• South Africa 1.8
25 countries – 11 industrial and 14 developing
Courtesy of Steve Weller27
Courtesy of Steve Weller
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