过表达Dbtnbt基因提高中国红豆杉细胞的紫杉醇含量  

Overexpression of a 3'-N-Debenzoyltaxol N-benzoyltransferase Gene Promotes Taxol Yield in Taxus chinensis Cells

作　　者：张鹏[1,2] 李书涛[2] 付春华[2] 周蓬蓬[2] 宋发军[1,2] 余龙江[2] 

机构地区：[1]中南民族大学生命科学学院,武汉430074 [2]华中科技大学生命科学与技术学院资源生物与生物技术研究所,武汉430074

出　　处：《中国生物化学与分子生物学报》2014年第4期377-382,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.31370118;No.20906036);中国博士后科学基金(No.20100471183)~~

摘　　要：本文通过克隆3'-N-去苯甲酰紫杉醇N-苯甲酰转移酶(3'-N-debenzoyltaxol Nbenzoyltransferase,DBTNBT)基因Dbtnbt,构建其表达载体p1303-SDbtnbtN,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;转基因细胞中报告基因GusA-mgfp5正常表达;转基因细胞的Dbtnbt mRNA表达量是未转化细胞的1.33倍;转基因细胞的紫杉醇产量约为27.3μg/g,是未转化细胞的1.37倍.本研究结果表明,过表达Dbtnbt基因将中国红豆杉细胞的紫杉醇产量提高约37%.In this study, one gene encoding 3＇-N-debenzoyltaxol N-benzoyhransferase （DBTNBT）, one key enzyme involved in taxol biosynthesis, was cloned and constructed into an expression vector pl303- SDbtnbtN. The pl303-SDbtnbtN was transformed into Taxus chinensis cells with an Agrobacterium mediated transformation method, and the Dbtnbt transgenic cells were obtained after hygromycin screening for generations. PCR and Southern blot analyses showed that the transgenes at the T-DNA region were integrated into the host chromosomes. Western blot analysis showed that a reporter gene GusA-mgfp5 could be detected. Quantitative real-time PCR analysis showed that the Dbtnbt expression level was 1.33- fold higher than that in non-transformed cells. I-IPLC analysis showed that the taxol yield from the transgenic cells was 27.3 pμg/g （ taxol/ dry weight of cells）, which was about 1.37 times of the non transformed cells. Our results showed that overexpression of the Dbtnbt gene increased taxol yield by 37% in the transgenic T. chinensis ceils.

关 键 词：红豆杉 3’-N-去苯甲酰紫杉醇N-苯甲酰转移酶(DBTNBT) 过表达 紫杉醇 

分 类 号：Q93[生物学—微生物学] R73[医药卫生—肿瘤]