Transcript lecture 5
Arabidopsis Experiments Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen / PCR Genotyping (H+- ATPase Mutants) Arabidopsis 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, tobacco1500 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 *Yours Truly 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 First, Forward Genetics Experiment #1 Conditional Screen for Ethylene Insensitivity Ethylene Egyptians gassed figs in order to stimulate ripening, “the gaseous hormone” The ancient Chinese burned incense in closed rooms to enhance the ripening of pears. H2C = CH2 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. 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 CTR1, active CTR1 inhibits the triple response, With ethylene present, or the receptor “absent”, or the CTR1 or the gene mutated, the triple response is activated. no ethylene ethylene, ein, etr, etc, …no triple response. …or ctr mutant, …blocks pathway. active inactive ? erf: ethylene response factor. induces transcription, Tuesday’s Work Sterilizing/Planting Germinating 70% ETOH/0.1% Triton X Breaking Dormancy 95% ETOH H2O/Imbibition, Murishige and Skoog Media (MS), O2/Aeration, plant minimal medium 0.5x strength With ACC. Cold/Prechilling "stratification” Inducing Germination Light ACC: 1-aminocyclopropane-1-carboxylate Conditional Screen Grow on ACC, …in the dark (etiolated). Score for mutants, Not this Class. Transfer to 0.5X MS (Murisige and Skoog) media (-ACC), Grow in light. What Next? dominant Thought Experiments… Backcross to wild-type, what might the F1 and F2 tell us? Complementation tests? recessive Second, Reverse Genetics Experiment #2 PCR genotyping of a t-DNA mutant 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. 115 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. 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 (ClustalW --> Phylip: protdist, fitch) Gene Family Gene AHA1 AHA2 AHA3 AHA4 AHA5 AHA6 AHA7 AHA8 AHA9 AHA10 AHA11 AHA12 Location whole plant root cortex phloem root endode rmis whole plant anthers seeds hypoco tyl - Function ? ? ? nutrient uptake ? ? ? ? ? ? ? psuedogen e 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 HormonesOpines Agrobacterium Lab T-DNA Out: Ti genes, opine genes, In: DNA of choice. Selectable Markers Reporter Genes 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 Breaking Dormancy H2O/Imbibition, Surface Sterilize Seeds Plant on Nutrient Media Germinate O2/Aeration, Cold/Prechilling "scarification” 1. EMS Treated Seeds on MS/ACC media. Inducing Germination 2. aha3-1 on MS media. 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’--GCATGCATTAT 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ CTGATCGTGAC--5’ o 94 Synthesis ~1 minute/kb 72o Denature Step ~30 seconds PCR ~65o Annealing Step ~30 seconds 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ CTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAT 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAT 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ CTGATCGTGAC--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 Your gene amplified 3’ Reverse Genetic PCR Strategy Reaction: Product: 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 2x backcross to wildtype phenotype analysis Tt T-DNA Segregation T t T TT Tt t Tt tt F2 PCR Genotyping 5’ homozygote wt 5’ heterozygote homozygote mutant L t T L t T L t 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ T Genetic Analysis F2 Segregation T t T TT Tt t Tt tt 1:2:1 Not Lethal T t T TT Tt t Tt tt 1 wt : 2 het Lethal T t T TT Tt t Tt tt 1 wt : 1 het Gametophyte Lethal Midterm Review on Wednesday, 1 hour midterm, Look for Ethylene-Insensitive Mutants.