甲基茉莉酸响应的bHLH类转录因子在青蒿素生物合成中的调控机制研究

Artemisinin is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its important medical value, increasing artemisinin content has become a hot research field. Although, the transcriptional regulation of artemisinin biosynthesis has already become an effective method in plant metabolic engineering, the regulation mechanism of artemisinin biosynthesis and the regulation network are still largely unclear. As an important plant hormone, methyl jasmonate (MeJA) has been proved that can promote the biosynthesis of artemisinin. In this proposal, we will focus on studying the mechanism of methyl jasmonate-responsive bHLH transcription factors (TFs) that involved in regulating artemisinin biosynthesis. Based on the A. annua RNA-Seq database, the bHLH TFs which are induced by MeJA treatment and have certain co-expression profiles with artemisinin biosynthetic pathway genes (such as ADS, CYP71AV1, DBR2 and ALDH1) will be cloned as candidate genes for further analysis. Dual-Luciferase reports and Yeast One-Hybrid assays will be applied to investigate the activation and binding elements between bHLH TFs and the promoter of artemisinin biosynthetic pathway genes. Finally, the best candidate bHLH gene will be overexpressed and decreased in A. annua via Agrobacterium tumefaciens-mediated transformation, in order to analyze the expression level of genes involved in artemisinin biosynthetic pathway and corresponding metabolites in transgenic A. annua. Together, the researches above will clarify the mechanism of methyl jasmonate-responsive bHLH TFs regulating biosynthesis of artemisnin, which will enrich and improve the transcriptional regulation network of artemisinin biosynthesis. Finally, this project will provide effective candidate genes for plant metabolic engineering.

青蒿素是治疗疟疾的特效药，具有重要医用价值，因此提高青蒿中青蒿素含量已成为研究的热点。虽然在转录水平调控青蒿素生物合成已成为青蒿素代谢工程研究中的重要技术手段，但青蒿素生物合成转录调控的相关机理尚不清晰。研究发现甲基茉莉酸（MeJA）能够有效促进青蒿素的生物合成，本项目拟开展MeJA响应的bHLH类转录因子参与青蒿素生物合成的转录调控机制研究。以转录组测序数据为基础，全面分析并克隆受MeJA诱导且与青蒿素合成途径功能酶基因有协同表达特性的bHLH基因；再通过双荧光素酶系统和酵母单杂系统，筛选出能与功能酶基因启动子有激活/结合活性的bHLH基因；最后采用转基因方法在青蒿中过量表达和RNAi干扰表达候选基因，研究转候选基因对青蒿素合成途径上相关基因的表达和代谢产物合成的影响。最终阐明bHLH转录因子参与调控青蒿素生物合成的机理，丰富和完善现有转录调控网络，为青蒿素代谢工程研究提供优质候选基因。

青蒿素是治疗疟疾的特效药，具有重要医用价值，因此提高青蒿中青蒿素含量已成为研究的热点，但青蒿素生物合成的转录调控其内在分子机理尚不完全清晰。本项目以青蒿全基因组和转录组数据为依托，通过基因共表达分析方法，成功筛选获得2个bHLH类转录因子（AabHLH2和AabHLH3），经一系列分子生物学、生物化学、植物转基因等手段深入研究AabHLH2和AabHLH3调控青蒿中青蒿素生物合成的机理。重点分析了AabHLH2和AabHLH3转录因子对青蒿素合成途径关键酶基因启动子的结合能力和激活作用，发现AabHLH2和AabHLH3以负调控方式抑制关键酶基因启动子的活性。进一步通过稳定转化青蒿实验也证明了AabHLH2和AabHLH3负调控青蒿中青蒿素的生物合成过程。最后我们进一步研究发现，AabHLH2和AabHLH3其N端缺乏转录激活域(TAD)，因而不能与AaMED25相互作用，从而竞争性的抑制AaMYC2和下游靶基因的相互作用，从而表现出负调控下游基因的表达。综上，本研究首次报道了负调控青蒿素生物合成的bHLH类转录因子，丰富和完善了现有青蒿素生物合成转录调控网络，有利于我们深入了解植物次生代谢途径调控的复杂性，并为以基因编辑等新技术改良青蒿品质提供了优质候选靶基因。