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 *Yours Truly
Forward vs. Reverse Genetics …we’ll be using both approaches. 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. H 2 C = CH 2 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 CTR 1, active CTR 1 inhibits the triple response, With ethylene present, or the receptor “absent”, or the CTR 1 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,
Wedsnesday’s To Do, #1 ems-treated seeds 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 +/- 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. 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
Second, Reverse Genetics Experiment #2 PCR genotyping of a t-DNA mutant
Wednesday’s To DO, #2 t-DNA insertion mutants 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 With ACC. Cold/Prechilling "stratification” Inducing Germination Light
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.
PCR Genotyping I. T-DNA Insertion Mutants II. Hs Mt_DAN Hypervariable Region
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(1) for agro with T-DNA Agrobacterium infect plant, select(2) for plants with T-DNA …if the T-DNA lands in a gene, the gene is disrupted.
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
Mitochondrial DNA - 16, 569 bp, - multiple copies per mt, - 1000 mt per cell, - 37 genes; - 22 oxidative phosphorylation, - 13 t. RNA, - 2 r. RNA, - Mitochondrial Control Region.
Mitochondrial Control Region • control region, – single promoter on each strand initiates transcription, – ori, • D-loop, – replication loop topography, • hypervariable region, – mutation rate 10 x greater than genome.
Mitochondrial Control Region • Hair follicle DNA extraction, • PCR, • Sequencing (at Cold Spring Harbor), • Sequence analysis here at WWU. Link Out
This Week? “hypervariable” Wednesday (probably) Mt-DNA isolation/PCR (maybe Fri. ), analyze EMS mutants on ACC screen. Wasps? Friday (maybe, maybe Weds. ) Isolate aha 3 -1 DNA and PCR genotype? other aha 3 -1 activity, Wasps?
- Slides: 46