bHLH类转录因子SmMYC1、2、3调控丹参酚酸及丹参酮合成的功能解析

Jasmonates (JAs) act as conserved elicitors of plant secondary metabolism. JA perception triggers extensive transcriptional reprogramming leadingto regulate the synthesis of plant secondary product. Previous reports showed methyl-jasmonate (MeJA) stimulates tanshinone and phenolic acids related gene expression and accumulation of tanshinone and phenolic acids in Salvia miltiorrhiza. In this project, RNA sequencing provided basic data that showed that three bHLH transcription factor (SmMYC1，2，3) expression was significantly induced after methyl jasmonate treatment. Here, we will detect its expression pattern in different tissues and changes between WT and MeJA-inducible plants of S. miltiorrhiza. Furthermore, ectopic overexpression and RNA interference (RNAi)-mediate knockdown experiment together with HPLC method will be taken in order to characterize the their function related to biosynthesis of tanshinone and phenolic acids. In order to characterize how SmMYC1，2，3 regulate four genes encoded key enzymes, promoters of SmCPS, SmCYP76AH1, SmRAS and SmCYP98A14 will be isolated and analyzed on basis of genomic data of S. miltiorrhiza. Using Electrophoretic Mobility Shift (EMS) and Chromatin.Immunoprecipitation (ChIP) assays, the binding specificity of SmMYC2 to the E-box motif in their promoters will be confirmed. To demonstrate that SmMYC1，2，3 act as transcriptional gene, co-transfection assays will be performed. This study will provide a detailed understanding of MeJA-inducible expression of multiple genes inthe tanshinone and phenolic acids biosynthetic pathway.

转录因子作为全面调控药用植物次生代谢合成的关键调控因子越来越受到药用植物学家的关注。前期研究显示茉莉酸甲酯可以有效地刺激丹参中的丹参酚酸类及丹参酮类合成相关基因的升高表达及化合物的积累。本项目通过检测茉莉酸甲酯处理后的丹参转录组信息并基因组信息，克隆三个bHLH类转录因子即SmMYC1、SmMYC2和SmMYC3; 利用实时定量PCR超表达及RNAi干涉等方法分析SmMYC1、SmMYC2和SmMYC3 的基因功能，进一步利用HPLC检测转基因苗中丹参酚酸及丹参酮含量的变化；通过克隆丹参酚酸及丹参酮合成途径上的关键酶基因SmCPS1、SmCYP76AH1、SmRAS1和SmCYP98A14的启动子区域并分析其E-box基元；并结合凝胶阻滞、顺时荧光及免疫沉淀实验验SmMYC1、SmMYC2 SmMYC3蛋白与启动子区的结合活性。在此基础上认知他们调控丹参酚酸和丹参酮合成途径的的功能冗余性。

中药材作为传统中药中最重要的品种，由于其含有两类有效成份即丹参酮类二萜类和丹参酚酸类化合物。丹参酮和丹参酚酸的含量常常受到茉莉酸甲酯激素的诱导。其中经典的bHLH类基因MYC2是整个茉莉酸信号途径中的关键蛋白。本项研究我们共关注三个受到茉莉酸甲酯诱导的bHLH基因，并重点关注了其中两个SmMYC2a和SmMYC2b的功能，这两个基因都定位在细胞核中。我们发现在两个MYC2基因都可以和茉莉酸甲酯代谢过程中的SmJAZ1和SmJAZ2互作。通过RNAi干涉实验发现SmMYC2a和SmMYC2b可以影响着丹参酮途径和丹参酚酸途径合成过程中的多个基因的表达情况。同时我们发现SmMYC2b可以结合到SmCYP98A14的启动子区调控着丹参酚酸的合成途径，SmMYC2a可以结合到SmHCT6和SmCYP98A14启动子区。高效液相检测结果也显示二者调控着丹参酮和丹参酚酸的合成，在调控方向上有着一定的区别。