摘要
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 genes encoding the key enzymes involved in taxol biosynthesis is an effective way to promote taxol yield in Taxus cells. In this study, we cloned the C-13 phenylpropanoid side ehain-CoA acetyltransferase （BAPT） gene and constructed it into an expression vector p1303- SbaptN. Transformed by Agrobacterium-mediated transformation method, the obtained Bapt transgenic Taxus chinensis cells were screened with hygromycin for generations. PCR and Southern blot analyses showed that the transgene was integrated into the host chromosomes at the T-DNA region. Western blot analysis showed that a reporter gene GusA-mgfp5 could be detected. Quantitative real-time PCR analysis showed that the Bapt expression level was 1.26 times higher than that in non-transformed cells. HPLC analysis showed that the taxol yield from the transgenic cells was 37.4 μg/g （ taxol/dry weight of cells） , which was about 1.87 times of the non-transformed cells. These results showed that overexpression of a Bapt gene was an effective approach to promote taxol yield in T. chinensis cells.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2013年第6期549-554,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.20776058
No.20906036)
中国博士后科学基金(No.20100471183)
运城学院自然科学基金((No.CY2012004)~~
