丹参MYC2转录因子高效调控丹参有效成分生物合成的机制研究

Methyl jasmonate (MeJA) has been known as a vital plant signal molecule which effectively regulates the biosynthesis of bioactive compounds in Salvia miltiorrhiza. MYC2 is a master transcription factor (TF) in jasmonate (JA) pathway. Our previous studies indicate that MYC2 is a positive TF which regulates the biosynthesis of active compounds in S. miltiorrhiza. However, the specific genes that MYC2 activates are still unclear and the regulation mechanism remains poorly understood. Chromatin immunoprecipitation (ChIP) assay will be implemented to investigate interacting genes of SmMYC2. Bioinformatics analysis and RNA interference (RNAi)-mediated knockdown experiments will be conducted to investigate the roles the target genes played in S. miltiorrhiza. To detect the specific binding of MYC2 and target genes, the electrophoretic mobility shift assay (EMSA) will be carried out. Transient expression assay will also be conducted to explore the genes that MYC2 could transcriptionally activate. The regulatory mechanism of SmMYC2 on the biosynthesis of active compounds will be clarified and the JA messenger-MYC2-TFs-pathway genes-active compounds network will be constructed. The above work will definitely contribute to unraveling the regulation mechanism of JA on bioactive compounds in S. miltiorrhiza. It will also facilitate achieving high yield of effective compounds in S. miltiorrhiza through metabolic engineering strategy.

茉莉酸甲酯（MeJA）是重要的植物信号分子，能够高效调控丹参有效成分的生物合成。MYC2是茉莉酸（JA）信号途径中的关键转录因子。课题组前期研究发现，丹参MYC2是调控丹参有效成分生物合成的正调控转录因子，然而，MYC2调控的靶标基因仍不明确，调控机制也有待进一步研究。本项目拟用染色质免疫共沉淀（ChIP）方法筛选出可被MYC2识别和结合的基因，对筛选出来的目标基因进行生物信息学分析和体内功能验证。通过凝胶阻滞电泳（EMSA）技术考察MYC2与目标基因启动子的特异性结合情况，再用瞬时表达分析的方法确定MYC2对目标基因的转录调控作用，从而解析MYC2高效调控丹参有效成分生物合成的机制，构建JA-MYC2-转录因子-关键酶基因-有效成分调控网络。本研究可为阐明JA高效调控丹参有效成分生物合成的机制奠定分子基础，同时为在丹参中开展代谢工程研究提供理想的候选靶点。

丹参是一种重要的药用植物，也是一种药用模式植物。本课题组前期研究表明，SmMYC2b是调控丹参活性成分生物合成的关键转录因子。然而，其调控机制仍不清楚。在本研究中，我们发现SmMYC2b的过量表达激活了丹参酮途径，而且SmMYC2b主要调控丹参酮的特异性下游途径。另外，我们发现，鼠尾草酸（CA）上游基因（SmCPS1，SmKSL1，SmCYP76AH1，SmCYP76AH3和SmCYP76AK1）的表达显著增加，而CA的含量显著降低，提示我们CA可能是丹参酮的重要前体，其含量降低是由于大量向下游丹参酮转化。进一步的CA饲喂实验为上述假设提供了证据。我们还发现SmMYC2b在促进侧根发育方面与其他同系物具有相似的功能。由于丹参酮类成分主要分布在丹参根的表皮，SmMYC2b的过量表达增加了丹参酮的合成场所，进一步促进了丹参酮类成分的生物合成。综上所述，我们认为SmMYC2b通过促进侧根发育和促进丹参酮途径来促进丹参酮的生物合成。我们的研究提供了一种提高丹参酮含量的有效策略，同时也丰富了我们对丹参酮成分调控网络的认识。