北柴胡茉莉酸信号通路核心转录因子BcMYC2的克隆及其调控柴胡皂苷生物合成的功能研究

Saikosaponin a and d is an important triterpenoid active ingredient in Bupleuri Radix , it has a significant effect in hepatoprotective, anti-tumor and anti-depressant-like. However the content of saikosaponin a and d in Bupleuri Radix is too low to satisfy the demand of Bupleurum extract and its new drug research. In the early stage of the project, building up a culture system of Bupleurum chinense hairy roots, it was found that the induction of methyl jasmonate could obvious regulate the content of saikosaponin a and d, its regulation mechanism may be related to hormone-induced jasmonic acid signaling pathway. MYC2 is the core transcription factor of jasmonic acid signaling pathway, it has a "multi-point regulation" advantage to the terpenoid metabolic pathway. This project has passed Bupleurum chinese comparative transcriptomics research and screened 16 MYC2 gene fragments preliminarily, to be cloned the full-length BcMYC2 gene and constructed the over-expression vector to verify the regulation of the key enzyme gene expression in the biosynthesis pathway of saikosaponin. And then beginning the regulated experiment about exogenous jasmonic acid methyl ester induced hairy roots and simulated drought stress seedlings, purpose to construct a regulatory network of "signal substance-BcMYC2-key enzyme gene-saikosaponin" and investigate the regulatory mechanism of BcMYC2 on the biosynthesis pathway of saikosaponin. In order to lay the foundation about greatly improve the content of saikosaponin a and d in the hairy roots and clarify the regulation of its secondary metabolism basis.

柴胡皂苷a、d是中药柴胡的重要三萜类活性成分，具有显著的保肝、抗肿瘤和抗抑郁样作用。但柴胡药材中柴胡皂苷a、d含量低，难以满足柴胡提取物及其新药研发的需求。项目前期建立了北柴胡毛状根诱导体系，发现外源茉莉酸甲酯诱导可显著提高柴胡皂苷a、d含量，其调控机制可能与激素诱导下的茉莉酸信号通路有关。MYC2是茉莉酸信号通路的核心转录因子，对萜类代谢途径具有“多点调控”优势。本项目已通过北柴胡比较转录组学研究，初步筛选出16条MYC2基因片段，将克隆BcMYC2基因全长，并构建过表达载体验证对柴胡皂苷生物合成途径关键酶基因表达的调控功能。然后开展外源茉莉酸甲酯诱导毛状根与模拟干旱胁迫种苗等调控试验，构建“信号物质-BcMYC2-关键酶基因-柴胡皂苷”的调控网络，探讨BcMYC2对柴胡皂苷生物合成途径的调控机制。为大幅度提高毛状根中柴胡皂苷a、d含量并阐明其次生代谢调控机制奠定基础。

柴胡是中医临床常用大宗药材之一，其主要成分柴胡皂苷被证实具有抗炎、抗肿瘤、保肝等多种药理作用，疗效确切，具有十分广阔的开发、应用前景。但在天然植物中合成量少，化学合成成本高，故利用合成生物学手段寻找调控柴胡皂苷合成的关键因子，对提高目标产物的生物丰度具有重要的研究意义。.本项目通过分析北柴胡二代转录组数据，首次完成了北柴胡中6个MYC转录因子家族基因的克隆，对其进行多物种序列比对、结构域分析及系统进化分析确定4条MYC2转录因子；发现1条能够响应MeJA诱导并影响柴胡皂苷生物合成途径的MYC2转录因子，将其命名为BcMYC2；完成了过表达毛状根的遗传转化，验证BcMYC2过表达对柴胡皂苷及4个关键结构基因HMGR、IPPI、FPS、β-AS的调控功能；基于外源MeJA诱导与模拟干旱胁迫实验筛选出最适宜诱导浓度及时间，观察到BcMYC2受OPR及内源茉莉酸含量变化影响，发现BcMYC2与4个结构基因及柴胡皂苷丰度之间具有相似表达模式，证实MYC2可响应干旱胁迫的内源JA信号影响柴胡皂苷生物合成及其关键酶基因表达；构建“信号物质-BcMYC2-关键酶基因-柴胡皂苷”网络进行探讨并得出关键结论：北柴胡JA途径核心转录因子MYC2通过感受OPR及内源JA含量变化响应外源MeJA诱导及干旱胁迫效应，能够“多位点”调控下游关键酶基因表达进而影响柴胡皂苷生物合成途径。.本研究为药用植物北柴胡遗传改良及有效成分柴胡皂苷生产提供重要理论基础、基因资源与技术条件，研究结果进一步拓展了柴胡皂苷次生代谢调控网络，为今后柴胡皂苷扩大化生产提供了毛状根等可持续性药用资源，具有重要的理论价值与应用前景。