The Precise Regulation Of Exogenous Gene In Transgenic Plants And Security Transformation Platform

With the rapid development of molecular biology and biotechnology, a considerable progress has been acquired on technology of transformation during the last years. Presently the problem is no longer obtaining transgenic plants, but is how to produce fertile transgenic plants efficiently with high-safety and marker-free. Over the past 30 years, development of transformation method of plant genetic, including Agrobacterium-mediated transformation, gene gun (microprojectile bombardment), electroporation, and microinjection et al. As having the remarkable advantage over direct transformation methods, containing the merit of easy-to-operated, low-cost, low copies. Agrobacterium-mediated transformation has become the most commonly method of genetic modification.The upstream regulatory region of the gene of heat shock protein HSP81.1 was separated from Brassica napus genomic DNA and the gene of cytochrome enzyme P450 was sepatated from Arabidopsis thaliana genomic DNA, according to the homology-based method. Bioinformatics analysis has shown that the gene of HSP81.1 is 2169bp and 2186bp with P450. Plant expression vectors were constructed by ligating HSP81.1 and P450 with GUS gene, and then transferred into tobacco by mediation of Agrobacterium tumefaciens, respectively. A large number of transgenic tobacco plants were obtained. Histochemical analysis of T0 transgenic plants shown that the HSP 81.1 promoter can be induced to express by the heat treatment. The expression of GUS was different at different growth stage, and the promoter has the highest activity at the growth stage of 50 days, but the P450 promoter has no specific space, expressed in tobacco roots, stems and leaves.Recently, with the development of biotechnology, the safety of GMO was widespread concerned, the most controversial focus on selecting marker gene. For transgenic plants, the selection marker gene is no longer useful when the target gene transformants were obtained. However, the selection marker gene has potential bio-security risk in transgenic plants, for an instance, whether the herbicide-resistant gene could bring on the emergence of super-weeds, impacting the ecological balance; or whether the antibiotic marker genes could transfer into human and animal's micro-organisms, affecting the efficiency of antibiotic treatment. Accounting for the safety of GMO, the system of Cre/loxP site specific recombination were constructed to facilitate deletion of marker genes which is between two direct LoxPs after successfully transformed. Plant expression vectors were constructed by ligating HSP81.1 and CaMV35S with Cre gene, and ligating CaMV35S with GFP gene, respectively. The selectable marker is bar gene which is between in the same direction loxP sites. The three vectors were transferred into tobacco using the method of mediation of Agrobacterium tumefaciens. A large number of transgenic tobacco plants were obtained. Hybridized the transgenic tobacco containing Cre gene and loxP site, the marker-free transgenic tobacco plants will be obtained.However, the problems of GMO safety are not only focusing on the selectable marker gene, but also become super-weeds, and there may have potential impact on the relative species and the wild species. Because of the same or similar growing, flowering, flowers between genetically modified crops and normal crops, so have a good chance for gene flow. In addition, the majority of transgenic crops seeds are very difficult to distinguish with nomal crops, it was easy to mingle with the spread, it may be lead to unpredictable environmental risk. So the barnase gene was amplified from the genomic DNA of Brassica napus (MS8RF3), Plant expression vectors were constructed by ligating PNap and P450 with Barnase gene and transferred into tobacco and rapeseed using mediation of Agrobacterium tumefaciens, respectively. And the vector of transgenic pBILoxp2 PNap TAibarnase in tobacco have been obtained, but the vector of transgenic pBILoxp2 P450 TAibarnase and pC1300 PNap Barstar L Barnase in tobacco have no positive plant.