Arabidopsis Experiments Forward Genetic Screen Ethylene Insensitive Mutants
Arabidopsis Experiments Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen / PCR Genotyping (H+- ATPase Mutants)
Arabidopsis thaliana is the predominant model organism used by plant biologists today. Considered a “weed" in nature, this small mustard serves as an experimental subject for everything from root growth to flower development in the laboratory. Arabidopsis has gained prominence as a model organism for several reasons: Generation time. Seed to seed in about 42 days. Fecund. One Arabidopsis plant yields thousands of seeds. Genome size. 125 megabases. Maize has 5000 mb, tobacco 1500 mb. Diploid. Relatively simple genome. Tractable. Easily worked and amenable to genetic, molecular genetic, physiological and biochemical studies. Real plant. Roots, leaves, flowers, seeds, and a full component of physiological and biochemical processes.
Arabidopsis History Linnaeus
Forward vs. Reverse Genetics Treat thousands of organisms with a mutagen, - random mutagenesis, Identify an individual with a phenotype of interest, Forward Identify the gene. • Treat thousands of organisms with a mutagen (usually), – random mutagenesis, • Identify an individual with a genotype of interest, • Identify the phenotype. Reverse
Ethylene “the gaseous hormone” H 2 C = CH 2 Egyptians gassed figs in order to stimulate ripening, The ancient Chinese burned incense in closed rooms to enhance the ripening of pears. In 1864, gas leaks from street lights were observed to stunt plant growth, twist plants, and abnormally thicken stems Dimitry Neljubow (1901) showed that the active component was ethylene. R. Gane (1934) reported that plants synthesize ethylene.
Receptor enzyme-linked receptor …found first in bacteria, then in plants, now in most eukaryotes, including mammals. Two-component regulators.
Ethylene …promotes fruit ripening, Ethylene signals the transition from unripe to ripe fruits, cell wall components are broken down, starches and acids are broken down resulting in “sweetening” and aromatic compounds , pigmentation may also be induced.
ACC: 1 -aminocyclopropane-1 -carboxylate
Ethylene …promotes the “triple response”, …in etiolated seedlings, reduced stem elongation, thicker stem, horizontal growth, May provide the plant with “behavior” that will provide escape from soil impediments.
Ethylene …mutant analysis, wild type ein wild type ctr ein (ethylene present), ctr (ethylene absent), …ethylene insensitive. …constitutive triple response.
Ethylene Signal Transduction …negative regulation. Tricky Concept(s) In the absence of ethylene, the enzyme receptor activates CTR 1, active CTR 1 inhibits the triple response, With ethylene present, or the receptor “absent”, or the CTR 1 or the gene mutated, the triple response is activated.
no ethylene, ein, etr, etc, …no triple response. …or ctr mutant, …blocks pathway. active inactive ? erf: ethylene response factor. induces transcription,
Friday’s Work Sterilizing/Planting Germinating 70% ETOH/0. 1% Triton X Breaking Dormancy 95% ETOH H 2 O/Imbibition, Murishige and Skoog Media (MS), O 2/Aeration, plant minimal medium 0. 5 x strength Cold/Prechilling "stratification” Inducing Germination Light
Conditional Screen Grow on ACC, …in the dark (etiolated). Score for mutants, Transfer to 0. 5 X MS (Murisige and Skoog) media (-ACC), Grow in light.
What Next? dominant Thought Experiments… Backcross to wild-type, what might the F 1 and F 2 tell us? Complementation tests? recessive
Proton Pumps in planta Pollen tip growth Anthers cell elongation Stems transport; sucrose hormones Arabidopsis Leaves stomata (gas exchange) sucrose transport Roots root hair growth mineral uptake Embryo/Seeds loading
H+ (protons) ATP synthase Transporters - carriers, - channels ATP hydrolase (ATPase) Adapted from Biochemistry and Molecular Biology of Plants, pp. 1
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.
Arabidopsis Experiments Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen / PCR Genotyping (H+- ATPase Mutants)
Proton Pumps in planta Pollen tip growth Anthers cell elongation Stems transport; sucrose hormones Arabidopsis Leaves stomata (gas exchange) sucrose transport Roots root hair growth mineral uptake Embryo/Seeds loading
Phylogenetic Family Tree Arabidopsis H+-ATPase (Clustal. W --> Phylip: protdist, fitch) Gene Family Baxter et al. , Plant Physiol, 123, (2003
Reverse Genetics Functional Genomics Gene DNA Sequence Gene Disruption Phenotype Analysis Function Mutate DNA Sequence Genetically Link Development Physiology Cell Biology
Nature Ti-Plasmid T-DNA Plant Cells Hormones. Opines Agrobacterium Lab T-DNA Out: Ti genes, opine genes, In: DNA of choice. Selectable Markers Reporter Genes
Agrobacterium tumefaciens Ti Plasmid (Tumor inducing) Mother Nature wt plant chromosome hormone genes (i. e. auxins) Ti Plasmid (from agro) opaline virulence genes nopaline neoplastic transformation opaline, nopaline virulence genes hormone genes Agro food
T-DNA (Transfer DNA) Laboratory Construct T-DNA selection genes virulence genes …can put other genes. transform, select for agro with T-DNA Agrobacterium infect plant, select for plants with T-DNA …if the T-DNA lands in a gene, the gene is disrupted.
Germination To Do Ø Breaking Dormancy Ø H 2 O/Imbibition, Surface Sterilize Seeds Plant on Nutrient Media Germinate 1. EMS Treated Seeds on MS/ACC media. 2. aha 3 -1 on MS media. Ø O 2/Aeration, Ø Cold/Prechilling "scarification” Ø Inducing Germination Ø Light
Probability of Finding an Insert in a Specific Gene p = 1 -(1 -f)n p = probability of insertion event f = 1 -(Genome/Size of Gene) n = number of T-DNA inserts thousands of inserts
Knockology Plants/Pools DNA/Pools
Set-Up DNA Pooling Maintain lines as pools of seed. Seeds (9) Germinate and grow seeds in liquid culture. Seedlings (225) Extract DNA, DNA (225) Super Pool DNA, 1 2 3 4 5 PCR Screen 6 … 30 Super Pools (2025)
5’--GCATTAT 5’--GCATTAGGCTACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGATCGTGAC--5’ 5’--GCATTAGGCTACATCGACTAGCACTG-3’ 5’--GCATTAGGCTACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGATCGTGAC--5’ 3’--CGTAATACGATGTAGCTGATCGTGAC--5’ 94 Synthesis ~1 minute/kb 72 o o Denature Step ~30 seconds PCR ~65 o Annealing Step ~30 seconds 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG-3’ CTGATCGTGAC--5’ 5’--GCATGCATTAT 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
PCR Strategy 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. Reaction: Product: 5’ Your gene amplified 3’
Reverse Genetic PCR Strategy Reaction: Product: none. T-DNA
PCR Screens for Mutants
PCR Strategy Reaction: T-DNA Product: Reaction: Product: T-DNA
Find the Plant You are ~here
T-DNA Mutants tagged seed line Genetic Analysis tt x TT (wt) isolate homozygous mutant 2 x backcross to wildtype phenotype analysis Tt T-DNA Segregation T t T TT Tt tt F 2
PCR Genotyping 5’ homozygote wt 5’ L t T L t 3’ 3’ 5’ 3’ heterozygote homozygote mutant 5’ 3’ T
Genetic Analysis F 2 Segregation T t T TT Tt tt 1 : 2 : 1 Not Lethal T t T TT Tt tt 1 wt : 2 het Lethal T t T TT Tt tt 1 wt : 1 het Gametophyte Lethal
Midterm Review on Wednesday, 1 hour midterm, Look for Ethylene-Insensitive Mutants.
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