Site-specific Recombination System Set Up In Brassica Napus

Site-specific Recombination System Technology was an technology that could create heredity transform to living things in the level of genes or chromosomes through the precise cutting and reconnecting of DNA specific sequences. Site-specific Recombination System played an important role in many areas of transgenenic plants such as fixed-point integration of genes,gene deletion,multiple-gene plant expression vector construction multiple-gene superposition and genetic breeding.The study constructed two expression vectors using Green Fluorescent Protein as reporter genes to create two site-specific recombination systems of Cre/loxP and FLP/FRT in Brassica napus based on the two systems.In the process of transgenic seedlings,we tried to find out the optimal concentration of kanamycin resistance screening with Agrobacterium tumefacinens dip method plants and succeeded transforming tweleve z1 and three Cer40 carrier positive seedlings through the plant tissure culture prosesses of co-culture,delay culture,screening culture and rooting culture.In the study,we also tried to find out a system of explants into seedlings about the extinction of positive seedlings as the result of microbiological contamination and transplant failure in the prosess then solved the problem and succeeded obtaining lots of aftergrowth applying the system on scarce seedlings.In the process of seedling detection,we found kanamycin resistence gene in cole genome through ordinary PCR and estimated them transgenic plants primarily.The analysis of PCR-southern blot based on the probe of CRE enzyme was within the anticipation and showed the expression carrier had integrated cole chromosomes.We also detected the expression of CRE gene in the level of RNA and gained the difference of each plant expression volume by means of semiquantitative method. Not only did it demonstrate the successful expression of site-specific recombination systems in Brassica napus.it also played the solid foundation for the future experiment.The key of the study lies in constructing two site-specific recombination systems of Cre/loxP and FLP/FRT in Brassica napus and detecting the expression of positive seedlings,laying the important foundation for gene fixed-point integration and multiple genes superposition in coles.The result of this study is of great importantance to the experiment in the future.