Download - 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.