过表达HDR和ADS基因对青蒿素生物合成的影响  

Enhancement of artemisinin biosynthesis in transgenic Artemisia annua L. by overexpressed HDR and ADS genes

作　　者：王亚雄[1] 龙世平[1] 曾利霞 向礼恩[1] 林智[1] 陈敏[3] 廖志华[1] 

机构地区：[1]西南大学生命科学学院三峡库区生态环境教育部重点实验室,重庆400715 [2]四川省眉山第一中学,四川眉山620010 [3]西南大学药学院,重庆400715

出　　处：《药学学报》2014年第9期1346-1352,共7页Acta Pharmaceutica Sinica

基　　金：国家高技术研究发展计划(863计划)资助项目(2011AA100605);国家自然科学基金资助项目(31070266)

摘　　要：青蒿素是从草本药用植物黄花蒿(Artemisia annua L.)中分离提取的一种含有过氧桥基团结构的新型倍半萜内酯,它是世界卫生组织推荐治疗脑型疟疾和抗氯喹恶性疟疾的特效药物。本研究采用过表达青蒿素生物合成途径中关键酶基因的方法,培育出青蒿素含量提高的转基因黄花蒿。采用根癌农杆菌介导遗传转化方法,潮霉素抗性筛选获得同时转化HDR和ADS两个基因的转基因黄花蒿;通过基因组PCR检测确认获得了6个共转化HDR和ADS基因的转基因株系。实时荧光定量PCR检测基因表达量,结果表明转基因株系中HDR和ADS基因表达量均高于非转基因植株(仅ah3株系ADS基因表达量没有显著性提高);HPLC测定青蒿素含量结果表明转基因株系中青蒿素含量均高于非转基因植株。青蒿素含量最高的转基因株系ah70中青蒿素含量为非转基因对照的3.48倍。本研究实验结果表明,过表达HDR和ADS基因在黄花蒿的代谢途径中具有提高青蒿素含量的作用,为进一步利用代谢工程技术培育青蒿素高产转基因黄花蒿提供了良好基础。Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. （Qinghao）. Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control （except ah3 in which the expression level of ADS showed no significant difference compared with control）; and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level thannon-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin.

关 键 词：黄花蒿 青蒿素 羟甲基丁烯基-4-磷酸还原酶 紫穗槐二烯合成酶 转基因 

分 类 号：R931[医药卫生—生药学]