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Arabidopsis Experiments

Arabidopsis Experiments. Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen / PCR Genotyping (H + - ATPase Mutants). Forward vs. Reverse Genetics. Treat thousands of organisms with a mutagen, - random mutagenesis,

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Arabidopsis Experiments

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  1. Arabidopsis Experiments Forward Genetic Screen (Ethylene Insensitive Mutants) Reverse Genetic Screen / PCR Genotyping (H+- ATPase Mutants)

  2. Forward vs. Reverse Genetics • Treat thousands of organisms with a mutagen, - random mutagenesis, • Identify an individual with a phenotype of interest, • Identify the gene. Forward • Treat thousands of organisms with a mutagen (usually), • random mutagenesis, • Identify an individual with a genotype of interest, • Identify the phenotype. Reverse

  3. Proton Pumps in planta Pollen tip growth Anthers cell elongation Stems transport; sucrose hormones Leaves stomata (gas exchange) sucrose transport Embryo/Seeds loading Roots root hair growth mineral uptake Arabidopsis

  4. H+ (protons) ATP synthase Transporters - carriers, - channels. ATP hydrolase (ATPase) Adapted from Biochemistry and Molecular Biology of Plants, pp. 115

  5. 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: 20,000 - 25,000.

  6. Proton Pumps in planta Pollen tip growth Anthers cell elongation Stems transport; sucrose hormones Leaves stomata (gas exchange) sucrose transport Embryo/Seeds loading Roots root hair growth mineral uptake Arabidopsis

  7. Phylogenetic Family Tree (ClustalW --> Phylip: protdist, fitch) Arabidopsis H+-ATPaseGene Family Baxter et al. , Plant Physiol, 123, (2003)

  8. Mutate DNA Sequence Genetically Link Reverse GeneticsFunctional Genomics Function Gene DNA Sequence Phenotype Analysis Gene Disruption Development Physiology Cell Biology

  9. T-DNA Ti-Plasmid Plant Cells Lab Selectable Markers Reporter Genes Genes Nature Hormones Opines Agrobacterium T-DNA Out: Ti genes, opine genes, In: DNA of choice.

  10. Agrobacterium tumefaciens Ti Plasmid (Tumor inducing) neoplastic transformation opaline, nopaline virulence genes hormone genes Mother Nature wt plant chromosome hormone genes (i.e. auxins) opaline virulence genes Ti Plasmid (from agro) Agro food nopaline

  11. transform, select for agro with T-DNA Agrobacterium infect plant, select for plants with T-DNA T-DNA (Transfer DNA) Laboratory selection genes …can put other genes. virulence genes Construct T-DNA …if the T-DNA lands in a gene, the gene is disrupted.

  12. Done Germination • Breaking Dormancy • H2O/Imbibition,  • O2/Aeration, • Cold/Prechilling "scarification”   • Inducing Germination • Light Surface Sterilize Seeds Plant on Nutrient Media Germinate 1. EMS Treated Seeds on MS/ACC media. 2. aha3-1 on MS media.

  13. p = probability of insertion event f = 1-(Genome/Size of Gene) n = number of T-DNA inserts Probability of Finding an Insert in a Specific Gene p = 1-(1-f)n thousands of inserts

  14. Krysan et al., 1999 Knockology Plants/Pools DNA/Pools

  15. Set-UpDNA 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, Super Pools (2025) 1 2 3 4 5 6 …30 PCR Screen

  16. 5’--GCATGCATTAT 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ CTGATCGTGAC--5’ CTGATCGTGAC--5’ 5’--GCATGCATTAT Denature Step ~30 seconds 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ Annealing Step ~30 seconds 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ CTGATCGTGAC--5’ 5’--GCATGCATTAT 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 94o Synthesis ~1 minute/kb 72o PCR ~65o

  17. 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: 5’ 3’ Your gene Product: Your gene amplified

  18. Product: Reverse Genetic PCR Strategy Reaction: Product: none. T-DNA Reaction:

  19. PCR Screens for Mutants

  20. T-DNA Reaction: T-DNA Reaction: Product: Product: PCR Strategy

  21. Find the Plant You are ~here

  22. T-DNA Mutants Genetic Analysis TT Tt Tt tt T t 2x T-DNA Segregation T t F2 tagged seed line tt x TT (wt) isolate homozygous mutant Tt backcross to wildtype phenotype analysis

  23. L t T homozygote wt L t T heterozygote 5’ 5’ 5’ 3’ 3’ 3’ L t T 5’ 5’ 5’ 3’ 3’ 3’ homozygote mutant PCR Genotyping

  24. TT TT TT Tt Tt Tt T T t t T t Tt Tt Tt tt tt tt T T T t t t 1 : 2 : 1 1 wt : 2 het 1 wt : 1 het Not Lethal Lethal Gametophyte Lethal Genetic AnalysisF2 Segregation

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