WRKY转录因子调控贯叶连翘褪黑素生物合成的机制研究

贯叶连翘提取物被广泛应用于轻至中度抑郁症患者的临床治疗。褪黑素是贯叶连翘中一种重要的生物活性成分，不仅可以诱发抗抑郁效应，而且可以保护植物免受氧化胁迫。但褪黑素在植物中表达水平极低，且受上游转录因子的调控机制尚不明确。基于此，我们在前期工作中首次建立起稳定的贯叶连翘遗传转化体系，其次构建高质量的全基因组数据库，并结合转录组数据，通过多组学联合分析、共表达模式、启动子作用元件分析筛选到一条很有可能参与调控褪黑素合成的转录因子HpWRKY75。本研究拟通过构建贯叶连翘HpWRKY75的过表达和敲除植株，测定褪黑素代谢途径上关键酶基因的表达丰度及次生代谢产物的含量，以阐明HpWRKY75在褪黑素生物合成中的关键作用；利用EMSA、酵母单杂交、Dual-Luciferase、ChIP等方法探究HpWRKY75对褪黑素代谢途径靶基因的调控机制，为利用代谢调控手段提高贯叶连翘中褪黑素含量奠定基础。

Hypericum perforatum extract is widely used in the clinical treatment of mild to moderate depression. Melatonin is an important bioactive component in H. perforatum. It can not only induce antidepressant effect, but also protect plants from oxidative stress as a plant growth regulator. Although melatonin biosynthesis pathway has been deeply studied, its extremely low plant expression level and the regulation mechanism of upstream transcription factors is not clear. Based on this, we established a stable genetic transformation system for H. perforatum for the first time, and then constructed a high-quality genome-wide database. Combined with transcriptome data, we screened a transcription factor HpWRKY75 which is likely to participate in the regulation of melatonin synthesis through Muti-omics, co-expression pattern and promoter element analysis. In order to elucidate the key role of HpWRKY75 in melatonin synthesis, the overexpression and knockout plants of H. perforatum were constructed, and the content of melatonin metabolic pathway related compounds and the expression abundance of key enzyme genes were determined. EMSA, Yeast one hybrid, Dual-Luciferase, CHIP and other methods were used to explore the regulatory mechanism of HpWRKY75 on melatonin metabolic pathway target genes. This study will lay a foundation for the use of metabolic regulation methods to improve the content of melatonin in H. perforatum.