GmWRKY30转录因子响应茉莉酸信号参与调控秦艽中龙胆苦苷生物合成机制的研究

Gentiana macrophylla is one of the most important traditional Chinese medicines. The main active components are secoiridiod glycosides with the highest proportion of gentiopicroside. At present, the research and development of "qinlongkusu" powder injection have completed the phase III clinical trial, in which gentiopicroside is the pharmacodynamic component, and it is a class a new drug for the treatment of hepatitis b, with broad application prospects. Using secondary metabolic engineering method to increase the content of gentiopicroside can relieve the pressure of G. macrophylla resources and solve the problem of insufficient supply of raw materials in the future market. However, the mechanism of biosynthesis and accumulation of gentiopicroside in G. macrophylla is unknown, which limits the use of metabolic engineering regulation methods in resource development. Based on this, Methyl jasmonate (MeJA) as a strong inducer to promote the synthesis of gentiopicroside in our research. Combined with transcriptome data, we found a transcription factor GmWRKY30 that is involved in regulating the content of gentiopicroside in response to JA signal. In this study, the RNAi and overexpression vectors of GmWRKY30 were constructed, and transgenic plants of G. macrophylla were obtained by agrobacterium-mediated genetic transformation. By comparing the changes in the content of gentiopicroside and the expression quantity of the key enzyme genes, the role of GmWRKY30 in the synthesis of gentiopicrin will be clarified. Then we cloned the promoter region of the key enzyme gene, and identified the GmWRKY30 binding target with the promoter region by using EMSA and yeast monohybrid technology. The transcriptional regulation effect of GmWRKY30 on enzyme genes was clarified by Dual Luciferase assay. So as to elucidate the regulatory mechanism of GmWRKY30 on target genes of the metabolic pathway of gentiopicroside. This study will lay a foundation for the use of metabolic regulation methods to improve the effective composition of G. macrophylla.

秦艽的有效成分是以龙胆苦苷为主的裂环环烯醚萜苷类化合物，以龙胆苦苷为药效组分治疗乙肝一类新药“秦龙苦素”粉针剂的研发已完成了III期临床试验，应用前景广阔。利用次生代谢工程方法提高龙胆苦苷含量，可以缓解秦艽资源压力，解决未来市场原料供给不足的问题。但龙胆苦苷生物合成积累机制不明，限制了该方法在秦艽资源开发中的使用。基于此，我们以MeJA作为促进龙胆苦苷合成的强诱导剂为突破口，发现了响应JA信号参与调控龙胆苦苷含量的转录因子GmWRKY30。在此基础上，本研究通过构建秦艽GmWRKY30的干涉和过表达植株，通过对裂环环烯醚萜苷类化合物含量的测定，以及龙胆苦苷合成途径关键酶基因的表达丰度，阐明GmWRKY30在龙胆苦苷合成的关键作用；利用EMSA、酵母单杂、Dual-Luciferase方法阐明GmWRKY30对龙胆苦苷代谢途径靶基因的调控机制，为利用代谢调控手段提高秦艽中有效成分含量奠定基础

秦艽的有效成分是以龙胆苦苷为主的裂环环烯醚萜苷类化合物，而龙胆苦苷积累及调控机制不明，阻碍了龙胆苦苷生物合成研究的进展。前期以MeJA作为促进龙胆苦苷合成的强诱导剂为突破口，发现了WRKY转录因子家族能够响应JA信号参与调控龙胆苦苷含量的生物合成过程。为了阐明调控机制，本课题采用瞬时过表达和干涉的方法，发现WRKY家族中的WRKY1和WRKY30能够显著调控龙胆苦苷的含量。采用VIGS分别干涉WRKY1、WRKY30后可显著降低龙胆苦苷含量的积累，瞬时过表达WRKY1龙胆苦苷含量显著提高，初步说明WRKY家族成员对秦艽裂环环烯醚萜苷类化合物的合成具有重要的调控作用。并利用DAP-seq技术挖掘秦艽全基因组范围的WRKY1和WRKY30可结合靶基因，WRKY1可结合位点为51559个peak，注释到与启动子区域结合的靶基因为563个，GO富集分析显示主要参与丝氨酸/苏氨酸蛋白激酶的活性，细菌响应等。KEGG富集分析显示主要富集通路为α-亚麻酸的代谢途径、泛醌等萜醌生物合成等。WRKY30可结合位点为25个peak，注释到与启动子区域结合的靶基因为10个，GO富集分析显示主要参与光合作用，醌结合位点以及NADH脱氢酶活性等。KEGG富集分析显示主要富集通路为RNA聚合酶，光合作用等。应用酵母双杂交技术对WRKY1互作蛋白进行筛选，其可以与E3泛素蛋白连接酶，脱落酸受体PYL8等互作。结合目前的研究工作，我们推测当秦艽受外源诱导子处理后，植株开启响应机制，WRKY1通过响应ABA信号通路，进而结合于SLS、MK等目标靶基因，通过调控龙胆苦苷代谢通路关键酶基因从而调控次生代谢产物的合成和积累。而WRKY30似乎更多的是参与光合作用，进而调控次生代谢产物的合成和积累。本研究工作对进一步深化WRKY1和WRKY30调控龙胆苦苷生物合成机制的解析奠定了坚实的基础。