CwJAZ6响应茉莉酸信号调控温郁金β-榄香烯生物合成的分子机制研究

The β-elemene is an efficient anti-tumor drug isolated from genuine herb Curcuma wenyujin. The extremely low content and difficulty in chemical synthesis of β-elemene restrict its clinical application. Improving the existing varieties to increase the accumulation of β-elemene is an effective way to solve this problem. However, the theoretical basis is very weak at present. In previous study, we have verified that MeJA enhanced the accumulation of β-elemene in C. wenyujin, and JAZs as the core of JA signal pathway were induced by MeJA, particularly CwJAZ6, but the regulatory mechanism is not clear yet. We speculate that TFs repressed by JAZs are involved in transcriptional regulation of enzyme genes in β-elemene biosynthesis pathway under MeJA treatment. In this study, CwJAZ6 will be used as a bait to screen the interacting proteins (TFs) by yeast two-hybrid and pull-down-MS techniques. The interaction between TFs (CwJAZ6 interacting proteins) and the promoters of enzyme genes in β-elemene biosynthesis pathway will be verified. Finally, we will construct JA-CwJAZ6-TFs-Enzymes signal pathway to clarify the mechanism of JA-induced β-elemene accumulation through CwJAZ6 in C. wenyujin. These will lay foundation for generating new varieties containing more β-elemene.

温郁金为浙江地区道地药材，其活性成分榄香烯可用于肿瘤治疗，因产量低、含量少，难以大规模化学合成，导致市场产需缺口较大。改良品种提高榄香烯积累是有效解决这一问题的方法，然而温郁金的分子理论基础非常薄弱。我们研究发现外施茉莉酸甲酯显著提高了温郁金β-榄香烯的积累，同时诱导了茉莉酸（JA）信号通路核心蛋白JAZs的表达，其中CwJAZ6最为明显，但具体的调控机制尚不清楚。推测JAZ抑制的转录因子，响应JA信号后被激活，上调β-榄香烯合成途径酶基因的表达，调控β-榄香烯的生物合成。本研究拟利用酵母双杂交系统和体外pull-down联合质谱，筛选与CwJAZ6互作的转录因子（TFs）；阐明TFs与β-榄香烯合成途径酶基因的转录调控关系。最终完整解析温郁金JA-CwJAZ6-TFs-Enzymes信号途径，阐明CwJAZ6介导JA信号调控β-榄香烯生物合成的分子机制，为温郁金新品种创制奠定理论基础。

温郁金是我国浙江地区“浙八味”之一，已有上千年的临床用药历史。温郁金挥发油中活性成分榄香烯，经循证医学（EBM）系统评价证实抗癌作用强、抗瘤谱较广，可以透过血脑屏障，对于人类多种癌细胞都具有显著的抑杀作用，而且没有毒副作用，是我国唯一具有自主知识产权的小分子抗癌植物药。我们研究发现外施茉莉酸甲酯可以显著提高β-榄香烯的积累量，并通过三个层次的研究阐述其调控机制。首先利用高通量转录组测序系统分析MeJA处理后温郁金RNA水平的差异；其次全转录组鉴定CwJAZ家族基因20个，并分析其结构特征、表达模式、相互作用关系；最后筛选出CwJAZ的相互作用转录因子CwMYC2，验证了CwMYC2通过调控温郁金萜类合成途径酶基因的表达，从而增强了温郁金中榄香烯的积累，完整解析温郁金JA-CwJAZ-CwMYC2-Enzymes信号途径。