黄花蒿AP2/ERF基因家族新成员AaERF9调控青蒿素生物合成的功能研究

项目名称： 黄花蒿AP2/ERF基因家族新成员AaERF9调控青蒿素生物合成的功能研究

项目编号： No.31501367

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 食品科学、农学基础与作物学

项目作者： 田娜

作者单位： 湖南农业大学

项目金额： 20万元

中文摘要： 黄花蒿因含有抗疟和抗癌活性成分青蒿素而成为我国农民增收的重要来源。然而国外正在开展青蒿素的半合成研究，严重冲击我国黄花蒿资源优势。因此，通过加强青蒿素生物合成调控研究来加快黄花蒿品种改良速度已可不容缓。申请者前期研究综合消减杂交技术、cDNA-AFLP和iTRAQ定量蛋白质组学技术从青蒿素高含量品种中分离到一个新基因AaERF9，初步证明该基因可能参与青蒿素生物合成调控。本项目拟采用过表达和RNA干扰技术上调和下调目标基因的表达，采用LC-MS和GC-MS检测青蒿素及其前体合成的变化，进一步验证新基因的确切功能，并采用凝胶阻滞实验、瞬时表达和染色质免疫共沉淀分析从体外和体内分析AaERF9对青蒿素合成关键酶基因的激活作用，探讨AaERF9调控青蒿素生物合成的分子机理。本项目为定向培育黄花蒿新品种提供有我国自主知识产权的靶基因，对可持续性开发我国药用植物资源、提高农民收入有十分重要的意义。

中文关键词： 青蒿；品种改良；基因克隆；青蒿素；转录因子

英文摘要： Malaria is one of the most important parasitic diseases, affecting at least 300 million people a year globally, and the number is rising due to the global warming in recent years. Atemisinin, an endoperoxide-containing sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., which is a herb of the Asteraceae family that has been used for centuries for the treatment of fever and chills in China, is currently the best therapy against malraia. In addition, artemisinin proved effective against hepatitis B, schistosomiasis, several blood parasitic protozoans, and against a variety of cancer cell lines including breast cancer, human leukemia, colon, small-cell lung carcinomas, and drug-resistant cancers. Artemisinin demand has increased sharply since its importance for human health. Since A. annua is currently the only practical source of artemisinin, it is a very important source for income improvement of Chinese farmer. However, semisynthesis of artemisinin is underway, which will negatively impact Chinese crop farming of A. annua. Therefore, it is high time to carry out research on regulation of artemisnin biosynthesis in order to improve artemisinin level in A. annua. Our previous work isolated a novel transcription factor AaERF9 containing one AP2 domain from a new cultivar with high artemisinin content by combination of SSH, cDNA-ATLP and iTRAQ. And the previous investigation indicated that AaERF9 might be involved in regulation of artemisinin biosynthesis. The present proposed work aimed to identify the function of AaERF9 and to unclose the mechanism. Firstly, the AaERF9 would be overexpressed in tobacco containing ADS, CYP71AV1, DBR2 and ALDH1 expression vectors. And then the production of dihydroartemisinic acid or its derivatives would be determined by LC-MS and GC-MS. Secondly, AaERF9 gene would be knocked down in Artemisia annua by RNA interference, and the difference of artemisnin between the wild-type and transgenic plant would be compared for identification of gene function. Finally, in order to characterize how AaERF9 regulate genes in artemisinin biosynthetic pathway, promoters of ADS, CYP71AV1, DBR2 and ALDH1 would be isolated and used to address the co-acting with transcription factor AaERF9 through electrophoretic mobility shift (EMS), co-transfection assays and chromatin immunoprecipitation. The proposed work is very significant for application and basic research, which should provide theory support for stabilization of A. annua and should put more insight into the regulation mechanism of artemisinin biosynthesis. 

英文关键词： Artemisia annua L.;variety improvement;gene cloning;artemisinin;transcription factor