JAZ蛋白与转录因子互作调控丹参酮类物质合成的研究

Tanshinones, as the major bioactive components of Salvia miltiorrhiza, are a group of abietane-type diterpenes. We have found that methyl jasmonate (MeJA) as an elicitor could effectively enhance the tanshinone synthesis. JAZ (jasmonate-ZIM domain) proteins are repressors of transcription factors in jasmonate signal transduction pathway. We isolated 4 JAZ genes in S. miltiorrhiza, and overexpression of the SmJAZ8 gene resulted in lower sensitivity to MeJA and the same amount of transhinones accumulation in S. miltiorrhiza hairy root compared to control. For further studying the role of JAZ protein in tanshinone synthesis regulation, here our group plan to use transgenic approaches including overexpression and RNA interference (RNAi) of the target genes in S. miltiorrhiza to uncover the JAZ gene family roles in tanshinone synthesis regulation. At the same time, the yeast two-hybrid system, bimolecular fluorescence complementation (BiFC) and pull-down assay will be performed to identify the transcription factors that interact with our functional JAZ in S. miltiorrhiza. And then we will also use transgenic approaches to study the function of these transcription factors in tanshinone synthesis regulation in S. miltiorrhiza. Furthermore, we will combine transcriptomic and metabolomic analyses to elucidate the function of the transcription factor in tanshinone synthesis regulation, and utilize the chromatin immunoprecipitation sequencing (ChIP-seq) and ChIP-qPCR to study the site of action of transcription factor. This present work could be necessary to elucidate the signal pathway of tanshinone synthesis and boost the development of traditional Chinese medicine.

丹参药用活性成分丹参酮类物质是一类脂溶性二萜。我们前期研究表明茉莉酸甲酯作为一种诱导子促进丹参酮类物质合成。JAZ (茉莉酸ZIM结构域)蛋白作为茉莉酸信号途径中的抑制因子抑制相关转录因子活性，我们克隆4个丹参JAZ基因，其中，过表达SmJAZ8的丹参毛状根对茉莉酸信号的敏感性降低，且丹参酮类物质含量不被茉莉酸甲酯诱导上调。为进一步研究JAZ蛋白参与调控丹参酮类物质合成的机制。本项目拟通过过表达和RNAi转基因技术明确JAZ蛋白家族调控丹参酮类物质合成的作用；酵母双杂交、BiFC和Pull-down实验筛选并验证与JAZ互作的转录因子；过表达和RNAi转基因技术分析转录因子调控丹参酮类物质合成的作用；转录组与代谢组整合分析转录因子参与调控的作用机制；并通过ChIP-seq和ChIP-qPCR明确其作用位点。本项目的实施对揭示丹参酮类物质合成的信号调控机制以及促进中药产业的发展具有重要意义。

丹参主要药用活性成分包括脂溶性丹参酮类物质和水溶性酚酸类物质。茉莉酸甲酯作为一种诱导子促进丹参酮类和酚酸类物质合成。JAZ(茉莉酸ZIM结构域)蛋白作为茉莉酸信号途径中的抑制因子抑制相关转录因子活性。我们全基因组鉴定分析了丹参SmJAZ基因家族，克隆得到了9条SmJAZs基因的全长序列，分别命名为SmJAZ1、SmJAZ2、SmJAZ3、SmJAZ4、SmJAZ5、SmJAZ6、SmJAZ8、SmJAZ9和SmJAZ10。除了SmJAZ5/6，其他SmJAZs均可以形成同源二聚体，大部分SmJAZs可以形成异源二聚体。在茉莉酸甲酯诱导下，过表达SmJAZs均显著降低了毛状根中丹酚酸B的积累量，过表达SmJAZ1、SmJAZ2、SmJAZ5、SmJAZ6和SmJAZ9显著提高了毛状根中丹参酮的积累量，而过表达SmJAZ3、SmJAZ4和SmJAZ8降低了毛状根中丹参酮的积累量。此外，过表达单一SmJAZ显著影响了其他SmJAZs的表达。在MeJA处理下，SmJAZ8-RNAi毛状根中丹酚酸和丹参酮的含量均显著提高。RNA-Seq结果表明同时受JA和SmJAZ8调控的差异表达基因主要富集在初生代谢、胁迫响应以及转录调控途径上。通过酵母双杂交共计筛选到289个SmJAZ8互作蛋白｡其中编号8-6是一个MYC类bHLH转录因子，将其命名为SmbHLH128。通过双分子荧光互补技术验证 SmJAZ8 与筛选到的转录因子SmbHLH128确实存在互作关系。通过酵母双杂交验证了SmbHLH128 与 SmJAZ8 的相互作用区域在SmJAZ8蛋白的Jas结构域｡过表达SmbHLH128显著促进丹参毛状根中丹参酮类化合物的积累。酵母双杂交分析结果表明，除SmJAZ5外，其他SmJAZs可以和SmMYC2a、SmMYB39互作，SmJAZ1/2/3/4可以和SmMYC2b互作，SmJAZ1/2可以和SmPAP1互作。综上所述，丹参SmJAZs在调控丹参酮类和酚酸类物质合成过程中的存在多样性和冗余性，其分子机制为：①SmJAZs形成同源和异源二聚体；②SmJAZs相互之间在转录水平上存在正向和负向调控；③SmJAZs与丹参酮类和酚酸类物质正负调控转录因子存在互作关系。本项目的实施对揭示丹参酮类和酚酸类物质合成的信号调控机制以及促进中药产业的发展具有重要意义。