Arabidopsis Experiments Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen...
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ArabidopsisArabidopsis Experiments ExperimentsForward Genetic Screen (Ethylene Insensitive Mutants)Forward Genetic Screen (Ethylene Insensitive Mutants)
Reverse Genetic Screen / PCR Genotyping (HReverse Genetic Screen / PCR Genotyping (H++- ATPase Mutants)- ATPase Mutants)
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ArabidopsisArabidopsis
AArabidopsis thalianarabidopsis thaliana is the predominant model organism used by is the predominant model organism used by plant biologists today. Considered a “weed" in nature, this small plant biologists today. Considered a “weed" in nature, this small mustard serves as an experimental subject for everything from root mustard serves as an experimental subject for everything from root growth to flower development in the laboratory. growth to flower development in the laboratory. ArabidopsisArabidopsis has has gained prominence as a model organism for several reasons: gained prominence as a model organism for several reasons:
Generation timeGeneration time. Seed to seed in about 42 days.. Seed to seed in about 42 days. FecundFecund. One . One ArabidopsisArabidopsis plant yields thousands of seeds. plant yields thousands of seeds. Genome sizeGenome size. 125 . 125 mmegaegabbases. Maize has 5000 mb, tobacco1500 mb.ases. Maize has 5000 mb, tobacco1500 mb. DiploidDiploid. Relatively simple genome.. Relatively simple genome. TractableTractable. Easily worked and amenable to genetic, molecular genetic, . Easily worked and amenable to genetic, molecular genetic,
physiological and biochemical studies.physiological and biochemical studies. Real plantReal plant. Roots, leaves, flowers, seeds, and a full component of . Roots, leaves, flowers, seeds, and a full component of
physiological and biochemical processes. physiological and biochemical processes.
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ArabidopsisArabidopsis HistoryHistory
LinnaeusLinnaeus
Yours Truly
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Forward vs. Reverse Forward vs. Reverse GeneticsGenetics
…we’ll be using both approaches.…we’ll be using both approaches. Treat thousands of organisms with a mutagen,Treat thousands of organisms with a mutagen,
- random mutagenesis, - random mutagenesis,
Identify an Identify an individualindividual with a with a phenotypephenotype of interest, of interest,
Identify the gene. Identify the gene.
• Treat thousands of organisms with a mutagen (usually),Treat thousands of organisms with a mutagen (usually),
– random mutagenesis, random mutagenesis,
• Identify an Identify an individualindividual with a with a genotypegenotype of interest, of interest,
• Identify the phenotype. Identify the phenotype.
Forward
Reverse
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First, Forward GeneticsFirst, Forward Genetics
Experiment #1Experiment #1
Conditional ScreenConditional Screen for for Ethylene Ethylene InsensitivityInsensitivity
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EthyleneEthylene“the gaseous “the gaseous
hormone”hormone”
Egyptians gassed figs in Egyptians gassed figs in order to stimulate ripening, order to stimulate ripening,
The ancient Chinese burned The ancient Chinese burned incense in closed rooms to incense in closed rooms to enhance the ripening of enhance the ripening of pears.pears.
In 1864, gas leaks from street In 1864, gas leaks from street lights were observed to stunt lights were observed to stunt plant growth, twist plants, plant growth, twist plants, and abnormally thicken and abnormally thicken stems stems
Dimitry Neljubow (1901) Dimitry Neljubow (1901) showed that the active showed that the active component was ethylene. component was ethylene.
R. Gane (1934) reported that R. Gane (1934) reported that plants synthesize ethylene.plants synthesize ethylene.
HH22C = CHC = CH22
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Receptor Receptor enzyme-linked receptorenzyme-linked receptor
…found first in bacteria, then in plants, now in most eukaryotes, including mammals.
Two-component regulators.Two-component regulators.
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EthyleneEthylene…promotes fruit …promotes fruit
ripening,ripening,
Ethylene signals the Ethylene signals the transition from transition from unripe to ripe fruits,unripe to ripe fruits,
cell wall cell wall components are components are broken down,broken down,
starches and acids starches and acids are broken down are broken down resulting in resulting in “sweetening” and “sweetening” and aromatic aromatic compounds ,compounds ,
pigmentation may pigmentation may also be induced.also be induced.
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EthyleneEthylene…promotes the “triple …promotes the “triple
response”,response”,
……in etiolated seedlings,in etiolated seedlings,
reduced stem reduced stem elongation,elongation,
thicker stem, thicker stem,
horizontal growth,horizontal growth,
May provide the plant May provide the plant with “behavior” that with “behavior” that will provide escape will provide escape from soil from soil impediments.impediments.
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EthyleneEthylene…mutant analysis,…mutant analysis,
wild typewild type einein
ein ein (ethylene present),(ethylene present),
……eethylene thylene ininsensitive.sensitive.
wild typewild type
ctr ctr (ethylene absent),(ethylene absent),
……cconstitutiveonstitutive t triple riple rresponse.esponse.
ctrctr
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Ethylene Signal Ethylene Signal TransductionTransduction
…negative regulation.…negative regulation.
Tricky Concept(s)Tricky Concept(s)
In the absence of ethylene, the In the absence of ethylene, the enzyme receptor activates enzyme receptor activates CTR1,CTR1,
active CTR1 active CTR1 inhibitsinhibits the triple the triple response, response,
With ethylene present, or the With ethylene present, or the receptor “absent”, or the receptor “absent”, or the CTR1CTR1 gene mutated, gene mutated,
the triple response is activated.the triple response is activated.
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ein, etr*, etc,
…blocks pathway.
ethylene,
…or ctr mutant,
no ethylene
…no triple response.
active
inactive
induces transcription,erf: ethylene response
factor.
?
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Wedsnesday’s To Do, Wedsnesday’s To Do, #1#1
ems-treated seedsems-treated seeds
Sterilizing/PlantingSterilizing/Planting GerminatingGerminating
Breaking DormancyBreaking Dormancy
HH22O/Imbibition, O/Imbibition,
OO22/Aeration, /Aeration,
Cold/Prechilling Cold/Prechilling "stratification” "stratification”
Inducing GerminationInducing Germination
LightLight
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ACC: 1-aminocyclopropane-1-carboxylate
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Conditional ScreenConditional Screen
Grow on ACC,Grow on ACC,
……in the dark in the dark (etiolated).(etiolated).
Score for mutants,Score for mutants,
Transfer to 0.5X MS Transfer to 0.5X MS (Murisige and (Murisige and Skoog) media (-Skoog) media (-ACC),ACC),
Grow in light.Grow in light.
NotNotthis this Class.Class.
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What Next?What Next?
Backcross to wild-type,Backcross to wild-type,
what might the F1 and what might the F1 and F2 tell us?F2 tell us?
Complementation Complementation tests?tests?
dominantdominant
recessiverecessive
Thought Experiments…Thought Experiments…
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Second, Reverse Second, Reverse GeneticsGenetics
Experiment #2Experiment #2
PCR genotypingPCR genotyping of a of a t-DNA t-DNA mutantmutant
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Wednesday’s To DO, Wednesday’s To DO, #2#2
t-DNA insertion mutantst-DNA insertion mutants
Sterilizing/PlantingSterilizing/Planting GerminatingGerminating
Breaking DormancyBreaking Dormancy
HH22O/Imbibition, O/Imbibition,
OO22/Aeration, /Aeration,
Cold/Prechilling Cold/Prechilling "stratification” "stratification”
Inducing GerminationInducing Germination
LightLight
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Proton Pumps Proton Pumps in plantain planta
Stemstransport; sucrose hormones Leaves
stomata (gas exchange)sucrose transport
Antherscell elongation
Pollentip growth
Embryo/SeedsloadingRoots
root hair growthmineral uptake
Arabidopsis
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Adapted from Biochemistry and Molecular Biology of Plants, pp. 115
H+ (protons) ATP synthase
ATP hydrolase (ATPase)
Transporters
- carriers, - channels.
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Arabidopsis Arabidopsis GenomeGenome
~125 Mb (Megabases, million base pairs),~125 Mb (Megabases, million base pairs),
Rice: 420 Mb, Human: 3 Gb,Rice: 420 Mb, Human: 3 Gb,
25,498 genes from 11,000 gene families,25,498 genes from 11,000 gene families, Rice: 32,000 - 50,000, Human: 25,000 - 66,000.Rice: 32,000 - 50,000, Human: 25,000 - 66,000.
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PCR GenotypingPCR GenotypingI.I. T-DNA Insertion MutantsT-DNA Insertion MutantsII.II. Hs Mt_DAN Hypervariable RegionHs Mt_DAN Hypervariable Region
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Proton Pumps Proton Pumps in plantain planta
Stemstransport; sucrose hormones Leaves
stomata (gas exchange)sucrose transport
Antherscell elongation
Pollentip growth
Embryo/SeedsloadingRoots
root hair growthmineral uptake
Arabidopsis
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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)
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Reverse GeneticsReverse GeneticsFunctional GenomicsFunctional Genomics
Gene DNASequence
Gene Disruption PhenotypeAnalysis
Function
MutateDNA Sequence
DevelopmentPhysiology
Cell Biology
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Agrobacterium
Plant Cells
NatureTi-Plasmid T-DNA
HormonesOpines
Lab
Selectable MarkersReporter Genes
Genes
Out: Ti genes, opine genes,
In: DNA of choice.
T-DNA
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wtwtplantplantchromosomechromosome
Ti PlasmidTi Plasmid(from (from agroagro))
hormone genes (i.e. auxins)hormone genes (i.e. auxins)
opalineopaline
nopalinenopaline
virulencevirulencegenesgenes
virulencevirulencegenesgenes
hormone geneshormone genes
opaline, nopalineopaline, nopaline
neoplastic transformationneoplastic transformation
Agrobacterium tumefaciensAgrobacterium tumefaciensTi Plasmid (Ti Plasmid (TTumor umor iinducing)nducing) Mother NatureMother Nature
AgroAgrofoodfood
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Construct T-DNAConstruct T-DNA
selection genesselection genes
virulencevirulencegenesgenes
infect plant, infect plant, select(2)select(2) for plants with T-DNA for plants with T-DNA
T-DNA (T-DNA (TTransfer DNA)ransfer DNA)LaboratoryLaboratory
transform, transform, select(1)select(1) for for agroagro with T-DNA with T-DNA
AgrobacteriumAgrobacterium
……if the T-DNA lands in a gene, the gene is disrupted. if the T-DNA lands in a gene, the gene is disrupted.
……can put other genes.can put other genes.
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Probability of Finding an Insert in a Specific Probability of Finding an Insert in a Specific GeneGene
thousands of insertsthousands of inserts
p = 1-(1-f)p = 1-(1-f)nn
p = probability of insertion eventp = probability of insertion event
f = 1-(Genome/Size of Gene)f = 1-(Genome/Size of Gene)
n = number of T-DNA insertsn = number of T-DNA inserts
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KnockologyKnockology
Plants/Pools DNA/Pools
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Set-UpSet-UpDNA PoolingDNA 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
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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
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PCR StrategyPCR Strategy
5’ 3’
PPolymerase olymerase CChain hain RReaction (PCR),eaction (PCR),
with oligonucleotide primers with homology with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers.the DNA sequence between the primers.
Your geneReaction:
Product:Your gene amplified
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Reverse Genetic PCR StrategyReverse Genetic PCR Strategy
T-DNAReaction:
Product:
Reaction:
Product: none.
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PCR Screens for PCR Screens for MutantsMutants
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PCR StrategyPCR Strategy
T-DNAReaction:
Product:
T-DNAReaction:
Product:
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Find the Find the PlantPlant
You are ~hereYou are ~here
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T-DNA T-DNA MutantsMutants
Genetic Analysis Genetic Analysis
taggedseed line
taggedseed line
isolate homozygous
mutant
isolate homozygous
mutant
backcrossto wildtypebackcrossto wildtype
2x
phenotype analysis
phenotype analysis
tt x TT (wt)
Tt
T-DNASegregation
TT Tt
Tt tt
T t
T
t
F2
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PCR GenotypingPCR Genotyping
L t T
5’ 3’
5’ 3’heterozygote
L t T
5’ 3’
5’ 3’homozygotewt
L t T
5’ 3’
5’ 3’
homozygotemutant
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Genetic AnalysisGenetic AnalysisF2 Segregation F2 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
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Mitochondrial DNAMitochondrial DNA
- 16, 569 bp,16, 569 bp,
- multiple copies per mt,multiple copies per mt,
- 100 - 1000 mt per cell,100 - 1000 mt per cell,
- 37 genes;37 genes;- 22 oxidative phosphorylation,22 oxidative phosphorylation,- 13 tRNA, 13 tRNA, - 2 rRNA,2 rRNA,
- Mitochondrial Control Region.Mitochondrial Control Region.
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Mitochondrial Control Region
• control region, – single promoter on each strand
initiates transcription,– ori,
• D-loop,– replication loop topography,
• hypervariable region,– mutation rate 10x greater than
genome.
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Mitochondrial Control Region
• Hair follicle DNA extraction,
• PCR,
• Sequencing (at Cold Spring Harbor),
• Sequence analysis here at WWU.
Link Out
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This Week?This Week?“hypervariable”“hypervariable”
Wednesday (probably)Wednesday (probably) Mt-DNA isolation/PCR (maybe Fri.),Mt-DNA isolation/PCR (maybe Fri.), analyze EMS mutants on ACC screen.analyze EMS mutants on ACC screen. Wasps?Wasps?
Friday (maybe, maybe Weds.)Friday (maybe, maybe Weds.) Isolate aha3-1 DNA and PCR genotype?Isolate aha3-1 DNA and PCR genotype? other aha3-1 activity,other aha3-1 activity, Wasps?Wasps?