茶树类黄酮下游途径代谢酶复合体的鉴定及互作机理

Tea plants (Camellia sinensis) contains an abundant flavonoid compounds. Catechins (flavan-3-ols) are the major components of flavonoids in the leaves. By searching genome database, we found that most of the genes involved in flavonoid pathway were multigene family. The complexity of flavonoid compounds and involved genes reflects the diversity of flavonoid biosynthesis pathway. With metabolic engineering technology, our previous study indicated a regulatory machine between the key enzymes, including F3H, DFR, and LAR proteins. In this study, we propose that multienzyme complexes operated in the flavonoid downstream pathway through protein-protein interaction, which catalyze key branch-point reaction of flavonoid biosythasis. The flavonoid biosythasis pathway of tea plants can be accurately regulated through controlling the key gene expressions. In order to discover the regulatory mechanism of flavonoid biosythosis in tea plants, we will screen differentially expressed genes involved in branch-point of flavonoid downstream pathway. And, we will confirm the interaction of membrane P450s (including F3’H and F3’5’H) with the proteins encoded by the selected genes by yeast two-hybrid, CoIP, and BiFC methods. The project, being completed as planed, would advance our understanding the regulatory mechanism of flavonoid biosynthesis, and would provide molecular basis for metabolic engineering of flavonoids.

茶树富含以儿茶素为主的多种类黄酮化合物，且该合成途径关键基因主要以多基因家族形式存在的。类黄酮物质和功能基因的复杂性印证了茶树类黄酮代谢途径的多样性和高效性。利用基因代谢工程技术，课题组初步印证了该途径下游关键酶(F3H、DFR和LAR)之间存在相互作用。本项目提出以下科学设想：(1).茶树类黄酮下游代谢通路可能以结构蛋白复合体形式存在，以实现儿茶素、黄酮醇、原花青素等合成的相对独立性；(2).茶树通过精确调控代谢酶复合体相关基因表达，以实现合成途径的多样性和高效性。为此，本项目拟在明确不同类黄酮代谢支路候选基因基础上，以锚定P450蛋白(包括F3’H和F3’5’H)为中心，利用酵母双杂、Co-IP和BiFC技术，开展相关类黄酮代谢途径下游互作蛋白的挖掘，阐明其互作机理。本项目顺利开展有望揭示茶树类黄酮物质生物合成途径多样性、高效性的分子机理，为类黄酮物质的代谢工程的研究奠定理论基础。

根据前期研究，课题组提出茶树体内可能存在类黄酮高效合成的代谢酶复合体。通过课题组成员的紧密合作，基本完成了课题任务。首先，课题组克隆了11条茶树类黄酮代谢关键基因；其次，课题组以P450蛋白(C4H、F3’H以及F3’5’H)为诱饵蛋白，通过茶树酵母文库筛选，获得了可能的桥梁蛋白；最后，利用酵母双杂、CO-IP和BiFC等技术，明确了茶树体内存在以锚定蛋白(C4H、F3’H以及F3’5’H)为中心的代谢酶复合体。本课题揭示了茶树儿茶素高效合成的分子机制，为类黄酮物质代谢工程奠定了理论基础。