南开大学 基因操作原理_第十二章.ppt

STEP I Helper plasmid mobilizes itself into 2nd E. coli strain containing intermediate vector. E. coli IV colE1 ori E. coli helper ori bom + E. coli helper IV STEP II Helper plasmid mobilizes itself and the intermediate plasmid into Agrobacterium. E. coli helper IV pGV3850 disarmed Ti plasmid A. tumefaciens Ti oriV + can not replicate A. tumefaciens pGV3850 disarmed Ti plasmid Ti oriV helper IV BINARY VECTOR SYSTEM modified Ti plasmid oriV vir T-DNA deleted 2 LB RB ori kanr polylinker 1 BINARY VECTOR LEAF DISC DICOTS vs. MONOCOTS 1. Wounded monocot tissues do not produce phenolics; pretreat with wound exudate from dicots 2. Ti Virulence Increase virulence by increasing the expression of virG and virE1 3. Wound sites in monocots tend to become lignified; use explants containing high proportion of actively dividing cells 4. nptII gene can not be used as selectable marker use hpt gene (hygromycinR) 5. Ri plasmid PLANT VIRUS VECTOR 1. Plant viruses are able to absorb to and introduce their nucleic acids into the plant cells； 2. Infected cells yield large amounts of virus, so recombinant viral vectors have the potential for high level transgene expression； 3. Viral infections are often systemic, consistent, rapid； 4. plant viruses neither integrate into nor pass through the germ line. Caulimoviruses --- dsDNA – CaMV Geminiviruses --- 2 ssDNA – maize streak virus RNA plant viruses --- TMV, PVX PEG DEAE-dextran Calcium phosphate Artificial lipids Dendrimers DIRECT GENE TRANSFER Physical Chemical Microinjection Pressure Electroporation Biolistics-particle bombardment Silica/carbon fibers Lazer mediated ELECTROPORATION Gene pulser II system (Bio-Rad) 35S-35S: double 35S promoter AMV: untranslated leader sequence SS: Arabidopsis signal sequence, induce target protein to ER HDEL: for retention of the protein in the lumen of the ER Nos-T: nos terminator EGFP: Enhanced Green Fluorescent Protein coding sequence Niedz, R. P., et al. Electroporation of embryogenic pr