茉莉酸通过JAZ8/bHLH14信号转导单元调控丹酚酸合成的分子机制研究

Salvianolic acids are major active ingredients of Salvia militorrihza which strongly induced by phytohormone jasmonates (JA), while the regulation mechanism is not clear. In the previous studies, we found that Jasmonate ZIM-domain protein 8 (JAZ8) is a critical regulatory factor in the JA-mediated salvianolic acid biosynthesis. Based on analyzing the transcriptome data, protein-protein interactions and correlation between genes and metabolites, we screened out transcriptional factor bHLH14. Given the potential interaction domain of bHLH14 with JAZ8, and its activation effects on key genes of salvianolic acid biosynthesis pathway, we thus hypothesize that JA-induced salvianolic acid biosynthesis is regulated by JAZ8/bHLH14 signaling module. In this proposal, we will firstly investigate the interaction between SmJAZ8 and bHLH14 by yeast-two-hybrid, GST pulldown and firefly luciferase complementation imaging (LCI) assays. Then we will elucidate the effects on bHLH14 on salvianolic acids biosynthesis, based on the transcriptiome and metabolome analysis of bHLH14 transgenic plants. Finally, we will validate the transcriptional activation effects of bHLH14 on the key genes in salvianolic acid biosynthesis pathway using DNase I footprinting, electrophoretic mobility shift assay (EMSA) and transient promoter activation assays. This project, if successful, will elucidate the underlying mechanisms of JA-induced salvianolic acid biosynthesis, and provide strategies for molecular breeding of medicinal plants.

丹酚酸是丹参重要活性成分，其生物合成受茉莉酸（JA）调控，但调控机制尚不明确。课题组前期研究发现，JA依赖性调控因子JAZ8参与JA诱导丹酚酸合成；分析JA处理JAZ8过表达株系转录组数据并结合蛋白互作及基因-代谢物相关性分析，发现bHLH14具有与JAZ8结合的结构基础、并对丹酚酸合成的关键酶基因有潜在转录调控效应。据此我们推测bHLH14与JAZ8形成JA信号转导单元介导茉莉酸诱导的丹酚酸积累。本项目拟通过酵母双杂交、荧光素酶互补技术和GST pulldown技术确证bHLH14和JAZ8的相互作用；比较bHLH14转化植株转录组与代谢谱明确其对酚酸合成的调控作用；利用DNA酶I足迹法、凝胶迁移率分析和启动子激活瞬时系统阐明bHLH14对酚酸合成途径上的关键酶基因的转录调控效应，从而揭示JA通过JAZ8-bHLH14诱导丹酚酸合成的分子机制。研究结果为药用植物分子育种提供依据。

茉莉酸能够显著调控丹参次生代谢物的积累，其中SmJAZ8蛋白作为核心成员参与调控，而SmbHLH14是潜在的靶标基因之一。首先SmbHLH14响应茉莉酸的信号，利用酵母双杂交和BIFC实验明确了SmbHLH14能与SmJAZ8相互作用，且SmbHLH14还受其他JAZ蛋白的调控。通过对SmbHLH14生物信息学分析，利用叶盘法获得过表达和敲除转基因株系，并对于该类植株次生代谢物含量测定，明确了SmbHLH14能够正向调控丹酚酸和丹参酮类的同时积累。通过相关代谢通路酶基因表达检测和启动子预测分析，结合酵母单杂交和荧光素酶互补检测实验确定了SmbHLH14正向调控酚酸类途径关键酶SmPAL1，SmC4H1和SmHPPR，丹参酮类关键酶 SmKSL1和SmCPS1的启动子区，从而影响基因转录，导致丹酚酸和丹参酮类次生代谢物含量变化。再结合和荧光素酶互补检测实验，可以得出结论SmJAZ8负向调控SmbHLH14从而改变影响下游靶基因结合启动子的能力，改变关键酶基因的表达，从而改变次生代谢的积累。本项目系统阐明JA通过JAZ8/bHLH14调控丹参酚酸类和丹参酮类活性成分合成的分子机制，同时利用茉莉酸信号途径解析了多个其他相关转录因子调控丹酚酸合成的机理，这些结果都为阐明丹参茉莉酸介导次生代谢过程的分子机制提供重要依据，为丹参高效、可行的改良提供思路及策略。