紫杉醇合成关键酶BAPT基因的克隆及原核表达  

Cloning and Prokaryotic Expression of BAPT Gene Related to Taxol Biosynthesis

作　　者：张志建[1,2] 郭佳玉[1,2] 张鹏[1,2] 徐盼盼[1,2] 刘博[1,2] 张燏[1,2] 付春华 余龙江[1,2] 

机构地区：[1]华中科技大学生命科学与技术学院资源生物学与生物技术研究所,湖北武汉430074 [2]华中科技大学生命科学与技术学院分子生物物理教育部重点实验室,湖北武汉430074

出　　处：《现代生物医学进展》2010年第16期3011-3014,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金面上项目(NSFC20776058);2006年教育部新世纪优秀人才支持计划(NCET-06-0646)

摘　　要：目的:克隆曼地亚红豆杉紫杉醇生物合成途径中的关键酶3-氨基-3-苯基丙酰转移酶(3-amino-3-phenylpropanoyltrans-ferase,BAPT)基因,并在大肠杆菌中表达。方法:首先提取曼地亚红豆杉细胞的总RNA,反转录获得其sscDNA,并以其为模板扩增BAPT酶的基因,然后将其与原核表达载体pET32a(+)连接,构建重组质粒pET32a(+)-BAPT,并将重组质粒转入E.coliBL21宿主中诱导表达。结果:扩增获得1338bp的BAPT基因序列,成功构建了原核表达质粒pET32a(+)-BAPT,并在E.coliBL21中实现高效表达。结论:BAPT基因表达产物的分子量约为51kDa,与预期大小一致。该研究为进一步分析曼地亚红豆杉BAPT酶的酶学特性奠定了基础。In this study, the total RNAs were isolated from Taxus x medium cells and were used to synthesize sscDNA by reverse transcript, and the fragments of the 3-amino-3-phenylpropanoyltransferase （BAPT） gene encoding one critical enzyme for taxol biosynthesis were amplified and were ligated with pET32a （＋） vector resulting to the recombinant plasmids pET32a （＋）-BAPT, which were transformed into E.coli BL21. The results showed that the BAPT gene consisted of 1338 bp, and the recombinant plasmids pET32a （＋）-BAPT were successfully constructed and expressed in E.coli BL2 l plysS, and the molecular weight of the BAPT gene was about 51 kDa, which was corresponding to the deduced size. These results lay a foundation for further researching the enzymatic characteristics of BAPT from Taxus x medium.

关 键 词：曼地亚红豆杉 紫杉醇 3-氨基-3-苯基丙酰转移酶基因 原核表达 

分 类 号：Q75[生物学—分子生物学] Q78