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过表达紫杉醇合成关键酶基因是提高红豆杉细胞紫杉醇产量的有效方法.本文通过构建C-l3苯丙素侧链CoA-乙酰转移酶(C-13 phenylpropanoid side chain-CoA acetyltransferase,BAPT)基因Bapt的表达载体p1303-SbaptN,采用根癌农杆菌介导法转化中国红豆杉细胞,经潮霉素抗性筛选获得转基因细胞系.转基因细胞的PCR和Southern印迹分析结果表明,T-DNA及其包含的基因与宿主细胞染色体成功整合;Western印迹结果表明,转基因细胞中报告基因GusA-mgfp5正常表达;QRT-PCR结果显示,转基因细胞的Bapt mRNA表达量是未转化细胞的1.26倍;HPLC检测结果显示,转基因细胞的紫杉醇产量为37.4μg/g,是未转化细胞的1.87倍.本研究结果表明,过表达Bapt基因提高了中国红豆杉细胞的紫杉醇产量.
Overexpression of paclitaxel synthesis key enzyme gene is an effective way to improve paclitaxel production in Taxus cell.By constructing C-13 phenylpropanoid side chain-CoA acetyltransferase (BAPT) gene Bapt The expression vector p1303-SbaptN was transformed into Taxus chinensis cells by Agrobacterium-mediated transformation of Agrobacterium tumefaciens, and the transgenic cell lines were screened by hygromycin resistance.PCR and Southern blot analysis of transgenic cells showed that T-DNA and its contained The result of Western blotting showed that the reporter gene GusA-mgfp5 was expressed normally in transgenic cells. The result of QRT-PCR showed that the expression of Bapt mRNA in transgenic cells was 1.26 times that of untransformed cells. The results of HPLC showed that, The yield of paclitaxel in transgenic cells was 37.4μg / g, which was 1.87 times that of untransformed cells.The results of this study indicate that overexpression of Bapt gene enhances paclitaxel production in Taxus chinensis cells.