青蒿紫穗槐二烯合酶基因的克隆与表达  

Cloning and Expression of Artemisia annua L. Amorpha-4,11-diene Synthase Gene

作　　者：黄瑛[1] 尹录录[2] 冯丽玲[2] 杨瑞仪[2] 杨雪芹[2] 曾庆平[2] 

机构地区：[1]广州军区广州总医院,广东广州510010 [2]广州中医药大学热带医学研究所,广东广州510405

出　　处：《广州中医药大学学报》2008年第1期68-73,共6页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：国家自然科学基金高技术探索项目(编号:30271591);面上项目(编号:30672614);国家中医药管理局科研基金(编号:02-03ZP43);广东省自然科学基金重点项目(编号:020799)

摘　　要：【目的】重建青蒿素合成代谢途径,以研制青蒿素高产微生物反应器。【方法】以低温预处理青蒿试管苗提取总RNA,采用逆转录聚合酶链反应(RT-PCR)扩增紫穗槐二烯合酶基因(ADS),并导入大肠杆菌中表达。【结果】青蒿ADScDNA测序结果已在GenBank注册。当ADScDNA与谷胱甘肽硫转移酶基因(GST)融合并在大肠杆菌表达后,其菌体裂解上清中可检测到谷胱甘肽硫转移酶(GST)活性,表明ADS基因已有可溶性表达。当裂解上清与法呢基焦磷酸(FDP)保温后,成功地检测到青蒿特有的倍半萜烯。【结论】青蒿ADS基因已在细菌体内实现功能性表达。Objective To reconstitute an anabolic pathway of artemisinin and to develop a microbial bioreactor for artemisinin.Methods Total RNA was extracted from the shoots of Artemisia annua L.cultured in vitro and pre-treated by low temperature,from which the amorpha-4,11-diene synthase gene（ADS） was amplified by RT-PCR and subsequently introduced into E.coli for overexpression.Results The sequence data of ADS cDNA was accessed in GenBank.When ADS cDNA was fused with the glutathion S-transferase gene（GST） and expressed in E.coli,the GST enzyme activity was detected in the bacterial lytic suspension,demonstrating the presence of soluble expression of ADS gene.After incubation with farnesyl diphosphate（FDP）,a sesquiterpene specific to Artemisia annua L.was successfully detected in the lytic suspension.Conclusion The functional expression of ADS gene cloned from Artemisia annua L.is achieved in the bacteria.

关 键 词：青蒿/化学 紫穗槐二烯合酶/分析 基因表达 

分 类 号：Q786[生物学—分子生物学]