The Cloning And Function Analysises That The Levansucrase Gene

Fructans is a kind of stored carbohydrate and locates in vacuole of higher plant .It depolymerizes ,then releases free fructn which can decrease water freezing-point of organization .The free fructn enhances drought - resistance and cold tolerance of the plants as vacuole osmotic buffer and cryoprotector .In this study , sacB form Bacillus subtilis was cloned by PCR . Homology of SacB and sequence published by Ebskampis 100% . pBI-sacB was constructed in which was regulated by CaMV35s promoter . pBI-sacB was transferred into tobacco via Argrobacterium-mediated method .Transgenic tobacco were obtained .After being transplanted ,transgenic tobacco appeared macular or necrosis on leaves .It was conferred that expression of sacB in cytoplasm disturbed normal physiological function of the plants and lead to harmful accumlation of fructans . In view of systhesis and accumlation of fructans in vacuole ,we constructed pBI-sp-sacB by fusing sacB with signal peptide sporamin guiding expression of sacB locating in vacuole .Normal transgenic tobacco were obtained by genetic transformation .Onions RNA was extracted with Trizol kit and obtained the sequence of cDNA of the onions SST by RT-PCR . Homology of sequence of SST and sequence published in Genbank is 99%. pBI-SST was constituted using the constitutive-expression vector pBI121. We transferred pBI-SST into tobacco via Argrobacterium-mediated method .Transgenic tobaccos were detected by molecular identification and active assay of fructan transferase .The result was that glucose level of transgenic tobacco glucose was obvious higher than that of non-transgenic tobacco ,which proved that SST integrated into plant genomic successfully and expressed exactly in transgenic tobacco .Cell membrane penetration was measured with PEG6000 stress on the leaves of transgenic tobacco .The result showed that relative conductivity of transgenic tobacco was lower than non-transgenic one .As primary research ,transgenic tobacco could enhance ability of plant-resistance-water .The research has potential practical value .Moreover , Function Analysis of SST from onions is not reported .The research lays a foundation for next function analysis .