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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:1526  下载量:727
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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