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

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.