Heat Inducible FLP Recominase Mediate Gene Excision From Transgenic Rapseed

With the research of plant genetic engineering going deeply, more and more people worry about the potential negative impacts of genetic modified plants on environment. Such as gene flowing, the effects on non-target life-form, overproducing of super weeds, generating of new virus, and biosafety of marker gene. In this research, A novel plant expression vector with gene deletion function was constructed by using heat shock promoter and site-specific recombination system, and transformed into tobacco. FLP recombinase, under the control of heat shock promoter Hsp18.2, catalyzed the foreign DNA( including knl, gus and npt II gene) flanked by two identical orientation loxP-frt sites to be deleted from genomics of transgenic tobacco. The special phenotype caused by knl gene in transgenic plants was recovered after heat shock treatment. This heat inducible FLP recombinase system was delivered into Rapseed (Brassica napus L), hoped to cultivate and select safe transgenic Rapseeds with high quality. The main results as follower:1 Gene deletion function of FLP recombinase system verification in tobacco The system (including 35S:kn1 expression element) was delivered into tobaccos,then heat shocked transgenic tobaccos. Histochemical stain, PCR and RT-PCR analyzing were performed. The results demonstrated that: After heat shocking, 64 lines of 171 lines were not tested GUS activity again, genomic DNA form transgenic lines without treatment and the leaves of transgenic lines 6,14 before treatment could be amplified flp gene fragment, while genomic DNA from normal leaves of line 6,14 after treatment could not be examined the specific fragment. It is concluded that: the foreign gene had been excised2 Optimization of rapeseed regeneration systems and genetic transformation systemsUsing hypocotyls, cotyledons and flower stems of three cultivars (Texuan 4, X1335-1, Huangzi 526) as explants to research the tissue culture and regeneration systems. The optimum differentiation medium was MS + 3mg/L 6-BA + 0.1 mg/L NAA Pre-culture and co-culture time, effects of genotype and different explants on resistance shoots frequency were researched. The optimum rooting medium was MS + 2.0mg/L IBA+ 0.05mg/L NAA, the rooting frequency reached to 93.9 %. Explants transformed with pBin-Ex-Hipt (containing 35S:ipt element) generated large number shoots. After transplanted in field, compared with wild type lines, the transgenic lines bloomed later for 72～80d, the flowering period was prolonged 20～25d, more branches, leaves crimped, color deeper, pods longer than control, and the anti-cold function increased3 Heat shocking treatment for transgenic rapeseeds and the effects of gene excisionAfter heat shocking treatment for 10 transgenic lines, GUS activities of lines 4,...