黑果枸杞花青素糖基化结构鉴定及其糖基转移酶功能解析

Anthocyanins are the major functional components in the fruit of Lycium ruthenicum Murr., an important medicinal plant. Glycosyltransferase plays an important role in multiple types of anthocyanins synthesis and accumulation. Up to now, the structures of anthocyanins and the types and functions of anthocyanins glycosyltransferase were remained unknown. This project intends to isolate anthocyanins using column chromatography, Prep-HPLC and further determine the structures of the glycosylated anthocyanins in the fruit of L. ruthenicum using NMR and MS. Thus, the type, site and number of the glycosylation of anthocyanins will be illustrated. Full-length cDNA of glycosyltransferase genes will be cloned by searching transcriptomic database and RACE method. The expression profile in different parts and stages of the fruits and the contents of anthocyanins will be detected by using real time PCR and HPLC-MS. The function of these genes will be determined by subcellular localization and the enzymatic assays in vitro. In the view of the foregoing, this project will reveal the structures and mechanism of glycosylation of anthocyanins in the fruits of L. ruthenicum. Ultimately, the project is not only to explore the scientific evaluation and accumulation mechanism of the anthocyanins in L. ruthenicum, but also to provide a theoretic guide for developing new varieties, increasing production and quality for L. ruthenicum.

药用植物黑果枸杞（Lycium ruthenicum Murr.）果实的主要药效成分之一是种类繁多的花青苷。糖基转移酶在催化合成种类多样的花青苷、促进花青素累积等方面起到重要作用。迄今为止，黑果枸杞花青苷的结构和糖基转移酶的种类、功能未知。本项目拟通过柱层析、Prep-HPLC等方法分离花青苷，结合NMR、MS等技术鉴定花青苷结构；明确黑果枸杞花青苷类化合物糖基化修饰类型、位点和数量等结构特点。通过转录组同源搜索、RACE等方法克隆糖基转移酶基因（UFGT）全长，利用实时定量PCR和HPLC-MS检测UFGT基因时空表达模式与花青苷的类型和含量；通过UFGT蛋白亚细胞定位、体外酶活检测等方法筛选、鉴定UFGT基因功能，旨在揭示黑果枸杞果实花青苷类物质结构及其糖基化修饰的机制。本项目不仅探索黑果枸杞花青苷类成分的科学评价指标及其累积机制，还将为黑果枸杞新品种培育、提高产量和品质提供理论指导。