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.