plant molecular genetics and genetic transformation hort 301 – plant physiology november 1, 2010...

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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