Arabidopsis Experiments Developmental Screen and Phase Changes Reverse Genetics PCR Genotyping.

Arabidopsis Experiments

Developmental Screen and Phase Changes

Reverse Genetics

PCR Genotyping

Phase Changes

flower development

juvenile to adultvegetative to reproductive

germination

zygote to embryo

gametophyte development

Phase Change Studies

• Genetic and molecular genetic approaches,

– isolate mutants that fail in some way to change phase properly,

– study genes, gene products and associated molecules, and resulting structures.

Forward vs. Reverse Genetics

• Treat thousands of organisms with a mutagen,

– random mutagenesis,

• Identify an individual with a phenotype of interest,

• Identify the gene.

• Treat thousands of organisms with a mutagen (usually),

– random mutagenesis,

• Identify an individual with a genotype of interest,

• Identify the phenotype.

Forward

Reverse

Proton Pumps in planta

Stemstransport; sucrose hormones Leaves

stomata (gas exchange)sucrose transport

Antherscell elongation

Pollentip growth

Embryo/SeedsloadingRoots

root hair growthmineral uptake

Arabidopsis

Adapted from Biochemistry and Molecular Biology of Plants, pp. 115

H+ (protons) ATP synthase

ATP hydrolase (ATPase)

Transporters

- carriers, - channels.

Arabidopsis Genome

~125 Mb (Megabases, million base pairs),

– Rice: 420 Mb, Human: 3 Gb,

25,498 genes from 11,000 gene families,– Rice: 32,000 - 50,000, Human: 25,000 - 66,000.

Gene Location FunctionAHA1 whole plant ?AHA2 root cortex ?AHA3 phloem ?AHA4 root endodermis nutrient uptakeAHA5 whole plant ?AHA6 - ?AHA7 - ?AHA8 - ?AHA9 anthers ?

AHA10 seeds ?AHA11 hypocotyl ?AHA12 - psuedogene

Arabidopsis H+-ATPase

Gene Family

Phylogenetic Family Tree(ClustalW --> Phylip: protdist, fitch)

Baxter et al. , Plant Physiol, 123, (2003)

Reverse GeneticsFunctional Genomics

Gene DNASequence

Gene Disruption PhenotypeAnalysis

Function

MutateDNA Sequence

DevelopmentPhysiology

Cell BiologyGenetically Link

Agrobacterium

Plant CellsNature

Ti-Plasmid T-DNA

Hormones Opines

Lab

Selectable MarkersReporter Genes

Genes

Out: Ti genes, opine genes,

In: DNA of choice.

T-DNA

wtplantchromosome

Ti Plasmid(from agro)

hormone genes (i.e. auxins)

opaline

nopaline

virulencegenes

virulencegenes

hormone genes

opaline, nopaline

neoplastic transformation

Agrobacterium tumefaciensTi Plasmid (Tumor inducing) Mother Nature

Agrofood

Construct T-DNA

selection genes

virulencegenes

infect plant, select for plants with T-DNA

T-DNA (Transfer DNA)Laboratory

transform, select for agro with T-DNA

Agrobacterium

…if the T-DNA lands in a gene, the gene is disrupted.

…can put other genes.

Probability of Finding an Insert in a Specific Gene

thousands of inserts

p = 1-(1-f)n

p = probability of insertion event

f = 1-(Genome/Size of Gene)

n = number of T-DNA inserts

Knockology

Plants/Pools DNA/Pools

Set-UpDNA Pooling

Seeds (9)

Seedlings

(225)

DNA (225)

1 2 3 4 5 6 …30SuperPools(2025)

Germinate and grow seeds in liquid culture.

Extract DNA,

Super Pool DNA,

Maintain lines as pools of seed.

PCR Screen

QuickTime™ and aCinepak decompressor

are needed to see this picture.

94o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Denature Step

~30 seconds

~65o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Annealing Step

~30 seconds

72o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

Synthesis~1 minute/kb

PCR

PCR Strategy

5’ 3’

• Polymerase Chain Reaction (PCR),

– with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers.

Your geneReaction:

Product:Your gene amplified

Reverse Genetic PCR Strategy

T-DNAReaction:

Product:

Reaction:

Product: none.

PCR Screens for Mutants

PCR Strategy

T-DNAReaction:

Product:

T-DNAReaction:

Product:

Find the Plant

You are ~here.

T-DNA Mutants Genetic Analysis

taggedseed line

taggedseed line

isolate homozygous

mutant

isolate homozygous

mutant

backcrossto wildtype

backcrossto wildtype

2x

phenotype analysis

phenotype analysis

tt x TT (wt)

Tt

T-DNASegregation

TT Tt

Tt tt

T t

T

t

F2

PCR Genotyping

L t T

5’ 3’

5’ 3’heterozygote

L t T

5’ 3’

5’ 3’homozygotewt

L t T5’ 3’

5’ 3’

homozygotemutant

Genetic AnalysisF2 Segregation

1 : 2 : 1

TT Tt

Tt tt

T t

T

t

Not Lethal

1 wt : 2 het

TT Tt

Tt tt

T t

T

t

Lethal

1 wt : 1 het

TT Tt

Tt tt

T t

T

t

GametophyteLethal

To Do

• Tuesday:

– Extract Plant DNA– PCR,– Continue Developmental Screen,

• Thursday:

– Run PCR fragments on gels,– Continue Developmental Screen,– Thin Developmental sceen plants.