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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.
ArabidopsisArabidopsis HistoryHistory
LinnaeusLinnaeus
Forward vs. Reverse Forward vs. Reverse GeneticsGenetics
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
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 In 1864, gas leaks from street lights were observed street lights were observed to stunt plant growth, twist to stunt plant growth, twist plants, and abnormally plants, and abnormally thicken stems thicken 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 R. Gane (1934) reported that plants synthesize that plants synthesize ethylene.ethylene.
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.
ACC: 1-aminocyclopropane-1-carboxylate
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.
……cconstitutiveonstitutive t triple riple rresponse.esponse.
ctrctr
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 or the gene mutated, or the gene mutated,
the triple response is activated.the triple response is activated.
Surface Sterilize Seeds Surface Sterilize Seeds Plant on Nutrient MediaPlant on Nutrient MediaGerminateGerminate
1. EMS Treated Seeds on 1. EMS Treated Seeds on MS/ACC media.MS/ACC media.
2. 2. aha3-1aha3-1 on MS media. on MS media.
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
KnockologyKnockology
Plants/Pools DNA/Pools
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
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 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.