Transformation Of Potato By RNAi Vector With Targeting Genes Of GBSS,SSⅢ,SSⅡ And Identification Of Transgentic Plant

Potato (Solanum tuberosum L.) owns a wide range of applications value in the food and chemical industries, furthermore lower-gelatinization temperature and faintish retrogradation of starch is also important reference. But the starch from most wildly cultivated potato cultivars hardly own better properties. With the further study of enzemy related to the biosynthesis and molecular mechanism of potato starch, it is possible that using technique of genetic engineering to regulate the key enzymatic gene expression for either lowing the gelatinization-temperature or rising faintish retrogradation of potato starch. In the present research, antisense-RNA technique was employed to regulate the expression of key genes in the biosynthesis potato starch to change the structure and components of starch granule, and further to develop processing potato lines with properties of lower-gelatinization temperature and better-stability of freeze thawing. The following are the main work we did:(1)The vectors of pART-GF and pART-PF, which initialized by partial of the GBSS and Patatin gene's promotor respectivly, were transformed into Agrobacterium Strain of LBA4404. And an engineered Agrobacterium Strain which contained target DNA site for further RNAi's conbination were maintained.(2)The stems and leaves from four species of potato(Solanum tuberosum.L) which named L3,WS1,XD and Z were invovled for explantation. An optimizaiton of concentration and proportion on growth hormone of NAA, 6-BA and GA3 were also performed based on culture of MS medium. Therefore an optimized system for potato's genetic transformation were preliminary established.(3)The species of L3,WS1,XD,Z were transferred by Agrobacterium tumefaciens of LBA4404 which have the expression vector. And a resistant plants were obtained by antibiotics resistance gene screening.(4) The GBSS gene's transcription levels in transgenic potato plants were analyzed by semi-quantitative RT-PCR, which indicated the content of transcriptase in transgenic potato was 32.5%, compared with that of in non-transgenic potato. Starch synthase activity in transgenic potato was lower than that in non-transgenic potato. Starch staining showed as blue-violet after staining the genetically modified potato plants, meanwhile the wild-type starch were grained showed as blue.