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T-DNA Mutagenesis Purpose: Determine gene function to produce better plants for society Mutagenesis Mutant: An organism that differs from the “normal” or wild type by one or more changes in its DNA sequence. Mutagenesis: Chemical or physical treatment that changes the nucleotide sequence of DNA. Can lead to changes in DNA sequence passed on to the next generation. Mutagenesis- creating mutants - Single nucleotide change G --> A Mutant Normal: Wild type ATTAGGCTACCGT TAATCCGATGGCA Mutagenesis -Or delete or add a nucleotide ATTAGACTACCGT TAATCTGATGGCA Mutagenesis- larger mutations - Delete a segment of DNA - delete many nucleotides X X Insert a segment of DNA = “Insertional” Insertion tagging • Principle: A DNA fragment (with a known sequence) is allowed to insert into the genome (when it lands in a gene, it usually causes a recessive, loss of function mutation). Insertion tagging • Advantages: – tags or marks the gene. – Provides a powerful way to identify or fish the gene out. • Disadvantages: – Cannot knock out essential genes. – Other redundant genes mask knock-out. – May disrupt non-functional sub-region of gene. Is it useful? • Highly and broadly useful • Applied to most organisms. • Mice, bacteria, yeast and plants have had their genes inactivated by knockouts. T-DNA Mutagenesis: A method of disrupting genes in plants with a “T-DNA” to “knock-out” gene function and activity. T-DNA = Transfer DNA a segment of DNA derived from the Ti plasmid contained inside the bacterium, Agrobacterium tumefaciens. “Agro” = plant pathogen Transferred from the bacterium to the plant. Randomly integrated into chromosomal sites in the nuclei. Agrobacterium tumefaciens - and Ti Plasmid Soil Bacterium infects plants through wounds & openings And causes crown gall tumors…. by expressing genes on a Ti plasmid - Tumor Inducing Plasmid Ti Plasmid • Contains genes for: Plant growth hormones - cytokinins and auxins. - stimulate undifferentiated growth Opine biosynthesis - food for Agrobact. Opine catabolism Acetosyringone receptors Plant wound produces acetosyringone Bacterial T-plasmid produces receptors for acetosyringone Bacteria is attracted to wound - receptor tells bacteria to swim to wound T-DNA is excised from Ti plasmid and integrates into plant genome. Genes on T-DNA are activated and stimulate cell proliferation. and Opine genes produce bacterial nutrients “Opines” IDEA: Ti- Plasmid, Tumor producing genes can be Replaced with other genes. New genes will be transferred! T-DNA region Left & right borders must be retained. Tumorproducing genes Opine catabolism Virulence region ORI Ti- Plasmid - delete genes for tumor and Agro nutrients T-DNA region X X XX Tumorproducing genes Opine catabolism Virulence region ORI Ti- Plasmid - delete genes for tumor and Agro nutrients T-DNA region New Gene Opine catabolism Virulence region ORI New foreign genes can be carried as passengers when the T-DNA integrates into plant genome. No tumors formed when auxin and cytokinin genes are replaced - plant has taken up T-DNA but no disease! = Disarmed Ti Plasmid What kind of genes can be added to T-DNA? - Any gene - Selectable marker Kanamycin Resistance Hygromycin R “ - reporter gene, marks cells to show they are transformed. Not always used. - genes for crop improvement, disease & insect resistance, new proteins, Vitamins, many possibilities Modified T-DNA for GFP Expression Left border HygR GFP Right border Plants will be hygromycin resistant and express green fluorescent protein. • Green fluorescent protein (GFP) From luminescent jellyfish Aequorea victoria. Produces green fluorescence under blue and UV light Redistribution of GFP-2SC in the Light Root Dark Light Root Hair cotyledon GFP-2SC moves from vacuole to ER and golgi, from Dark to Light Protoplasts: plants with cell walls removed. Modified T-DNA for Mutagenesis Left border KanR Right border Plants will be Kanamycin resistant. Might disrupt a gene or spacer DNA. Transformation with Disarmed Ti-plasmid in Agrobacterium - Mix Agro containing Ti-plasmid with: - Wounded leaf - Plant cells in culture - Floral dip under vacuum -plant cells or seeds on growth media containing selection antibiotic (i.e. Kan). -Only engineered plants grow Genome-wide insertional mutagenesis of Arabidopsis thaliana (2003) • Objective: create loss of function mutations for all genes. • Strategy: use T-DNA (with kanamycin-resistance gene as selectable marker) to generate collection of 150,000 T1 transformants. • > 225,000 independent T-DNA integration events thus far. Arabidopsis • Genome size = 125,000 kb; Average gene length = 2 kb • Random distribution of insertion events, predicts 96.6% probability of finding an insertion in an average gene • To determine the site of integration of each T-DNA, junction sequences were analyzed and 88,122 sites were proven to be at a single genomic location • Of the 29,454 annotated genes, 21,799 (74%) were hit. • Create a catalog and allow researchers to order seeds for their favorite gene disruption on-line. Not all genes can be knocked out. CNGC10 2000 bp T-DNA T1 generation - first generation after T-DNA insertion Single T-DNA insertion Distribution of T-DNAs showed hot spots (in gene-rich regions) and cold spots (in centromere and Peri-centromeric regions) T-DNA - heterozygous - 1 normal gene - 1 disrupted gene Obtaining Homozygous - 2 T-DNAs in same gene N T Heterozygous is self-pollinated N T N NN NT TN TT T Need homozygous - both copies knocked out T-DNA - Homozygous Screen for homozygotes by PCR using combinations of primers to the T-DNA and to the target gene to be knocked out PCR screen T-DNA mapping Gene 5’ T-DNA Gene 3’ No PCR product with this primer Non-perfect, but usable, results