何首乌二苯乙烯苷芪合酶的功能研究

Stilbene glycoside content is the main factor of the polygonum multiflorum quality. Stilbene glycoside forms through the corresponding biosynthetic pathway, in which stilbene synthase plays an important role. This research focuses on stilbene synthase, the key enzyme of the biosynthetic pathway of stilbene glycoside. The total RNA extraction, RT-PCR, heterologous expression are used to get the primary sequence of stilbene synthase; and then homology modeling, molecular docking, dynamic simulation are used to get the information of the interactions between stilbene synthase and the substrate,; and then the generation mechanism of stilbene glycoside after the binding of stilbene synthase and the substrate are speculated. Gene knockout technology is used to verify the speculation. The mechanism of biosynthetic pathway of stilbene glycoside in polygonum multiflorum will be detected by the experiments and simulations. The reason of the stilbene glycoside content variation in different polygonum multiflorum and the effect of stilbene synthase on polygonum multiflorum quality will be illuminated. Since currently stilbene glycoside content in polygonum multiflorum is of varying quality, we expect to find methods to stabilize high stilbene glycoside content, and ensure the high quality of polygonum multiflorum, and provide theoretical foundation to ensure the quality of traditional chinese medicine and to the identification.

二苯乙烯苷的含量是确定何首乌品质的主要指标，而芪合酶在二苯乙烯苷的生物合成途径中发挥着关键作用。本项目拟从影响二苯乙烯苷含量的关键酶-芪合酶入手，借助总RNA的提取、反转录PCR扩增、异源表达等方法得到芪合酶的一级序列；借鉴同源模建、分子对接、分子动力学模拟等分子模拟的理论、思路和技术手段得到芪合酶与底物结合模式的信息，进而推断何首乌中芪合酶与底物复合得到二苯乙烯苷的作用机制；再以基因定点敲除实验验证模拟得到芪合酶作用位点信息的真实性和可靠性。从实验和理论两个方面共同推测何首乌中芪合酶在二苯乙烯苷的生物合成途径的作用机制，以期揭示二苯乙烯苷含量差异的本质，同时分析芪合酶的作用对何首乌品质的影响。寻求稳定二苯乙烯苷含量，保证滇产何首乌药材品质的新思路和新方法，解决目前存在的何首乌中二苯乙烯苷含量差异大、产品质量不稳定的问题。为明确何首乌地道产区、稳定药材质量与药材品质提供科学的理论依据。

本项目从影响二苯乙烯苷含量的芪合酶入手，借助总RNA的提取、反转录PCR扩增、异源表达等方法得到芪合酶的一级序列；借鉴同源模建、分子对接、分子动力学模拟等分子模拟的理论、思路和技术手段得到芪合酶与底物结合模式的信息，再以基因定点敲除实验验证模拟的结果。从实验和理论两个方面共同得到何首乌中芪合酶在二苯乙烯苷的生物合成途径的关键性催化作用机制。在研究过程中，我们还对二苯乙烯苷类似的白藜芦醇和查尔酮进行了研究，合成了一系列新的白藜芦醇、查尔酮的衍生物并测定了体外抗炎活性和癌症抑制活性。