Cloning Of Chi And Glu Genes And Constructing Of Their Expression Vector Drived By Vascular-Specific Promoter And Transformation Into Cucumis Melo L

Cucumis melo L. is one of the most common fruit in the world. It is also people's favourite fruit for its special characters such as sweet-tasting, nutritious, valuable and so on. However, great loss was caused because most of Cucumis melo L. cultivars used for the production lack of resistance to disease or only have single resistance to disease,so the broad-spectrum resistance Cucumis melo L. cultivar is in great need. Tranditional Breeding methods is time consuming and inefficient, it is difficult to collect multiple residtance genes into a cultivar. As opposed to it, plant genetic engineering is a short-cut to improve a cultivar for its effiency and directness. In this study, the functional element of Populus vacular specific-expression promoter BSP was cloned from the genome DNA of Populus deltoids. Enzyme digestion and sequence analysis indicated:the length of the cloned fragementwas 860 bp and it contained regions and repeated sequences that were riched A/T bases . The cloned fragement shared 98.2ï¼… homology with the corresponding known sequence. GFP reporter gene drived by cloned BSP promoter, was introduced into tobacco through the partical gun transformation method, the transient expression of GFP proved the cloned promoter was active.Chi gene and Glu gene were cloned from plasimid pART27Tch and pBLGC by polymerase chain reaction (PCR) using artificial synthetic special primers respectively. Sequence analysis indicated:the cloned Chi gene was 1275bp,and the homology between this fragement and the corresponding reported sequence was 99.45ï¼…. The cloned Glu gene was 1088bp,the homology between this fragement and the corresponding reported sequence was 99.81ï¼….Consequently the Chi and Glu genes were inserted into pET28a(+) expression vector respectively, the N-terminal His-taq was introduced into the recom- binant enzyme. The recombinant protein was produced after induced by IPTG. The total proteins of positive and control bacteria were analyzed on a 15ï¼… polyacrylamide gel. Result indicated the molecular weight of expressed protein fit in theory very well. Additionally fungistasis experiment indicated the expressed proteins inhibited fungus strongly.Next by cutting off the aimed fragement BSP-Glu-terminator from plasmid pBIG and combining it with plasmid pBIC we constructed the plant expression vector pBIBCG of Chi gene and Glu gene, then intergrated it into agrobacterium LBA4404.Finally the interest genes were introduced into leaf and stem of Cucumis melo L. through Agrobacterium tumeformation-mediated transformation . Analyzing the transformation effects of different recipient material showed leaf of test-tube plantlet was better than stem for its high callus induction efficiency, strong ability to differentiation and regeneration and easy to obtain. Studies on transformed plants NO 2 and NO 5 showed Agrobacterium transformation efficiency was elated to the genotype of Cucumis melo L.. At present NO 2 transformed plant is propogating so as to assaying.