ANR与LAR在茶树表型儿茶素生物合成中的作用机制研究

The catechins with high level and multi-type in tea plant play an important role in enhancing the resistance of tea tree and improving tea beverage quality. It is generally believed that anthocyanin reductase (ANR) and leucoanthocyanidin reductase (LAR) are responsible for the synthesis of epicatechins and nonepicatechins, respectively. However, in previous research, we found ANR and LAR have new catalytic functions, that the direct product of ANR reaction in plant was a kind of flavan-3-ol carbocation, and LAR actually was involved in the biosynthesis of epicatechin. So how do ANR and LAR control the biosynthesis of epicatechins? This project was proposed to use tea, Arabidopsis and Lotus corniculatus as the experimental materials, in which the catechins are accumulated specifically, to study their recombinant LAR and ANR proteins, and the different catalytic pathways of them by enzymatic analysis. The relationship between the gene (LAR and ANR) expression profiles and the accumulation patterns of epicatechins in transgenic plants was studied by means of overexpression and CRISPR/Cas 9 gene editing technology using tobacco, Arabidopsis and tea hairy roots transformation systems. The aim of this project is to provide a theoretical basis for understanding the accumulation of high levels of epicatechins in tea plants, by clarifying the relationship between LAR/ANR and the biosynthesis of epicatechins.

茶树中高含量和多类型的儿茶素类物质对增强茶树抗性和提高茶饮料品质具有重要作用。普遍认为花青素还原酶(ANR)和无色花青素还原酶(LAR)分别负责表型与非表型儿茶素的合成。本项目前期研究发现，ANR与LAR具有新的催化功能，ANR在植物体内产物是一类黄烷-3-醇碳正离子，而LAR参与了表型儿茶素的生物合成。那么ANR与LAR是如何控制茶树表型儿茶素合成的？本项目立项拟以儿茶素类型特异性积累的茶树、拟南芥和百脉根为材料，以它们的LAR和ANR重组蛋白为研究对象，通过酶学特性分析，研究LAR和ANR不同催化途径与表型儿茶素积累的关系；借助烟草、拟南芥和茶树发根遗传转化体系，利用过表达和CRISPR/Cas 9基因编辑技术，研究LAR和ANR基因表达与转基因植株中表型儿茶类积累的关系。本项目旨在通过明确LAR和ANR与茶树表型儿茶素生物合成的关系，为解析茶树积累高含量儿茶素提供理论依据。

茶树叶片中富含茶多酚、咖啡碱和茶氨酸等多种次级代谢物质。作为茶树中主要次级代谢产物之一，儿茶素类化合物约占茶树中总多酚含量的70%，是赋予茶和多种饮料苦涩和收敛性品质特征的主要成分。根据 C环 C-2 和 C-3 位立体异构特点，儿茶素可分为表型儿茶素和非表型儿茶素。传统的类黄酮代谢途径认为花青素还原酶(ANR)和无色花青素还原酶(LAR)是负责表型与非表型儿茶素合成的关键酶，然而课题组前期发现ANR和LAR在参与表型儿茶素合成过程中具有不同以往的功能。.本项目以茶树、拟南芥和百脉根三种积累不同类型儿茶素的物种为材料，系统研究了LAR和ANR在植物体内和体外的功能，同时也对二氢黄酮醇-4-还原酶（DFR）的功能进行了更深入地研究，取得了以下研究结果：.（1）ANR在植物体内主要负责EC类碳正离子的合成，它可以作为PAs聚合过程中EC类型的延伸单元，但是ANR在体外可以催化花青素生成表型儿茶素单体；.（2）LAR作为一个关键的催化儿茶素合成的还原酶，其到底催化表型还是非表型儿茶素的生成，取决于它底物的构型：当底物是(2R, 3S)-trans-类型的化合物时，产物是非表型儿茶素，如C；当底物是(2R, 3R)-cis-类型的底物时，产物是表型儿茶素, 如EC；.（3）DFR催化二氢黄酮醇类底物生成无色花青素类，无色花青素类物质一方面可以被催化形成C类型单体儿茶素，也可以形成C类碳正离子，作为PAs聚合过程中C类型的延伸单元。.本项目的开展阐明了茶树中表型儿茶素合成的分子机理，也为解释茶树中高积累多酚类物质提供了理论依据。