丹参酮ⅡA合酶以及丹参2-ODD家族基因的功能研究

丹参（Salvia miltiorrhiza Bunge）是我国传统药用植物，以根和根茎入药，药理学表明丹参中丹参酮类化合物对治疗心脑血管疾病具有显著疗效，其中丹参酮ⅡA被广泛应用于临床。丹参酮类生物合成的上游途径已经被解析，多个CYP450家族蛋白被证明参与了丹参酮类化合物的氧化修饰，然而下游具有重要活性的丹参酮ⅡA和丹参酮I等化合物的合成途径还没有被解析。申请者在前期的研究中，通过对丹参不同组织转录组进行差异表达分析，确定了可能催化丹参酮类生物合成的2-ODD家族候选基因，进一步通过RNAi转化丹参发根，筛选到丹参酮ⅡA合酶的2-ODD家族候选基因。本项目拟通过原核表达、体外酶活鉴定、过表达/RNAi转基因和分子对接等技术来研究丹参酮ⅡA合酶及关键2-ODD蛋白的功能及其反应机制。本项目的实施，将为丹参酮类途径的解析和合成机制的阐明提供重要依据。

Salvia miltiorrhiza Bunge is a well-known traditional Chinese medicinal herb. As the major active secondary metabolites of S. miltiorrhiza, tanshinones mainly accumulated in roots and rhizome which are the important medical resources for the treatment of cardiovascular disease and cerebral vascular disease, and tanshinone ⅡA (TIIA) is one of the most important compounds and is widely used in clinical. Despite the upstream biosynthetic pathway of tanshinone is elicited and several CYP450s have been identified involving the modification of tanshinones, the biosynthetic pathway of TⅡA and tanshinone I (TI) has not been fully defined. Based on previous study, we have analysed RNA sequencing database from different tissues of S. miltiorrhiza. Combined with RNAi study, we have obtained a 2-ODD family candidate gene may be responsible for producing TⅡA. In this study, our group plan to assay the enzyme activity using Escherichia coli in vitro system, study their kinetics, and detect the contributions of the enzymes to the pathway through overexpression/RNAi. We will confirm the functions and activities of TⅡA synthase and the key 2-ODD enzyme by docking. The results of this present work could be necessary to elucidate the biosynthetic pathway of tanshinone, and provide referenced values for the metabolism illumination.