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

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.

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。

黄花蒿，又称青蒿，是我国重要的中药资源，在发展我国生物产业、提高农民收入等方面发挥了重要作用。黄花蒿有效成分青蒿素是一种抗疟特效药，由于全球气温变暖的原因，近年疟疾的发病率呈上升趋势，青蒿素的国际需求量越来越大。我国科学家屠呦呦因在青蒿素的发现和应用中的突出贡献而获得了诺贝尔奖，这是中国科学家首次获得该类大奖。此外，由于青蒿素还具有特异杀死多种癌细胞而对正常细胞无影响的特性，有望将其开发为高效、低毒、价廉、谱广的抗癌新药，具有广泛的应用前景。虽然化学合成青蒿素以及利用基因工程菌合成青蒿素前体物质取得了重要进展，但由于成本过高无法实现工业化生产。目前，青蒿素的商业化生产均是从黄花蒿植株中提取。因此，培育优良品种植株是降低青蒿素生产成本的主要手段。黄花蒿品种的有效改良依赖于对青蒿素生物合成调控机制的了解，但目前尚未完全清楚。本项目在前期研究发现了一个新的基因AaERF9可能调控青蒿素生物合成的基础之上，利用SMART-RACE技术扩增了AaERF9基因全长cDNA序列，并采用正、方向遗传方法验证了该基因的功能。此后，联合采用转录组学、蛋白组学、代谢组学以及凝胶阻滞实验、瞬时表达荧光检测和染色质免疫共沉淀分析等技术手段，深入分析了AaERF9调控青蒿素生物合成的分子机理，筛选了其调控对象，并对其调控基因进行了克隆与功能鉴定。结果表明，AaERF9通过促进青蒿素生物合成前体物质二氢青蒿酸的合成从而调控青蒿素在青蒿中的富集，并且发现 了AaERF9可调控黄花蒿中3个基因，即AaF3H、AaFLS和AaDBR3的表达，暗示了AaERF9可能通过调控黄花蒿中萜类化合物和黄酮类化合物生物合成途径中的碳流而改变青蒿素的含量。最后，对AaERF9调控的3个基因进行了克隆与功能验证。本项目的完成取得了重要的成功，已发表论文7篇，其中SCI收录5篇、核心期刊论文2篇，此外还有2篇论文待发表；已经授权国家发明专利5项；获得省科技进步三等奖1项。本研究结果为黄花蒿品种改良提供了新的途径，具有重要的学术和应用价值。