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.