Cloning The Genes Of Resisting Herbicide, Constructing Of Plant Expression Vector And Genetic Transformation Of Rape

Since the fact that adaptability and reproductive capacity to the environment of weed is more powerful than crop and traditional methods cannot satisfy manufactural needs, so chemistry herbicides are becoming the most useful ways to get rid of weed. With the application of the chemistry herbicide in the agriculture, especially the using of some new efficient herbicide, though the yield of crops has improved greatly, the function mechanism of herbicide influences the physiology and biochemical process of plants. When weeds are killed, the crop is hurted too. This has limited the application of herbicide .Via genetic engineering to introduce herbicide-resisting gene into the cultivar of crop, then the crop is offered the resistance to herbicide. The anti-herbicide crop is obtained by screening. This thchnology is important to improve production and quality of crops, and enlarge the application of herbicide.This study separated the rbcS promoter of RuBisCo small subunite and signal peptide coding sequence of chloroplast from genemotic DNA of pea via PCR. Sequence analysis indicated that the cloned fragment shared 98.41% homology with the reported sequence. It contained control element-TATA box and CAAT box, besides, it has the tissue-specific elements which act as an enhancer. Plant expression vector pBIRB-GFP was constructed by ligating the promoter and transport peptide with green fluorescent protein (GFP) gene, then transferred it into leaf of tobacco and epidermis cell of onion by microprojectile bombardment. The results indicated that this expression system has strengthened the transient expression of GFP. rbcS promoter and signal peptide coding sequence can achieve tissue-specific and light-regulated expression of interest gene, It offered a new way for high expression of foreign genes in the transgenic plant, and has important value in the theoretical research and production practices.We have cloned the Nitrile hydrolase gene (bxn) from klebsiella pneu moniae subspecies ozanae and cloned the bar gene from plasmid pBA. Sequence analysis showed they shared high homology with reported gene. Insert it into microorganism expression vector pET-28(a) respectively, then transformed it into E.coli BL21.Albumen molecules of 42kD and 24kD are obtained by IPTG induced, protein purification and SDS-PAGE electrophoresis. This indicates the molecular weight of expressed protein fit in theory value well, and bxn gene and bar gene areintact.rbcS promoter, bxn gene and bar gene are cut by restrict enzyme according to designation. And then combine them together, so the plant expression vector pBIBAH-CRBN are constructed.bxn gene and bar gene were transformed into Cytoledon petiole and aseptic cytoledon of 4-6 days of rape by microprojectile bombardment and Agrobacterium mediation. Rape callus was obtained by screening of Kanamycin. The transformed plant of rape has not been obtained yet.