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紫穗槐-4,11-二烯合酶基因克隆及其在大肠杆菌和酿酒酵母中的表达
发表时间：2010-06-15 浏览量：1956 下载量：918
| 全部作者： | 孔建强，沈君豪，黄勇，王 伟，程克棣，朱平 |
| 作者单位： | 中国医学科学院&北京协和医学院，卫生部天然药物生物合成重点实验室&中草药物质基础与资源利用教育部重点实验室 |
| 摘 要： | 从青蒿中克隆得到紫穗槐-4,11-二烯合酶（amorpha-4,11-diene synthase，ADS）基因，该基因长为1 641 bp，编码537个氨基酸。将该基因分别克隆到大肠杆菌（Escherichia codi）表达载体pET-32a(+)和酿酒酵母（Saccharomyces cerevisiae）表达载体pYeDP60/G中，构建能表达ADS基因的E.coli表达载体pETADS和酿酒酵母表达载体pYeDP60/G/ADS. 将pETADS导入E.codi BL21（DE3）中进行诱导表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western blot结果显示：该基因得到了表达。气相色谱-质谱联用仪（gas chromatography-mass spectrometer，GC-MS）对重组酶产物分析表明：法尼基焦磷酸（farnesyl pyrophosphate，FPP）被ADS环化形成了紫穗槐-4,11-二烯（amorpha-4,11-diene,AD）。将pYeDP60/G/ADS转入酿酒酵母W303-1B中，获得含ADS基因的酿酒酵母工程菌，该酵母工程菌的发酵产物经GC-MS检测，除了检测到紫穗槐-4,11-二烯外，还检测到了另外9种产物。 |
| 关 键 词： | 药物化学；青蒿；紫穗槐-4,11-二烯合酶；大肠杆菌；酿酒酵母；气相色谱-质谱联用仪 |
| Title： | Expression and functional characterization of amorpha-4,11-diene synthase in E.coli and S.cerevisiae |
| Author： | KONG Jianqiang, SHEN Junhao, HUANG Yong, WANG Wei, CHENG Kedi, ZHU Ping |
| Organization： | Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC & Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education of PRC |
| Abstract： | The amorpha-4,11-diene synthase (ADS) gene, which is 1 641 bp long and encodes 537 aa, was cloned from Artemisia annua L. The ADS gene was subcloned into the Escherichia coli and yeast expression vector pET-32a(+) and pYeDP60/G, respectively, resulting in the expression plasmid pETADS and pYeDP60/G/ADS. The pETADS was transported into E.coli BL21(DE3) and then induced to be expressed. The SDS-PAGE and Western blot results showed that the recombinant ADS was expressed successfully in E.coli. The soluble fraction was used to test the function of the recombinant enzyme. Amorpha-4,11-diene (AD) was detected by GC-MS with FPP as a substrate. The pYeDP60/G/ADS was introduced into Saccharomyces cerevisiae W303-1B to yield the engineered yeast containing ADS gene. GC-MS analysis showed that the engineered yeast produced amorpha-4,11-diene as a main product and other 9 trace sesquiterpene compounds as well. |
| Key words： | pharmaceutical chemistry; Artemisia annua L; amorpha-4,11-diene synthase; Escherichia coli; Saccharomyces cerevisiae; gas chromatography-mass spectrometer |
| 发表期数： | 2010年6月第11期 |
| 引用格式： | 孔建强，沈君豪，黄勇，等. 紫穗槐-4,11-二烯合酶基因克隆及其在大肠杆菌和酿酒酵母中的表达[J]. 中国科技论文在线精品论文，2010，3（11）：1094-1101. |
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