金柑类黄酮糖基转移酶基因功能分析及其调控网络构建

Fortunella hindsii is an important plant resource native to China, which has high food, medicinal, and ornamental value. Fortunella hindsii fruits are rich in flavonoids, and could be used as an idea research material for flavonoid biosynthetic and regulatory mechanisms. Based on transcriptome data mining, we isolated a gene encoding flavonoid UDP-glycosyltransferase (FrUDPG) and two relative transcriptional factors (FrMYB1 and FrMYB2) from Fortunella hindsii fruits. This research focuses on the biological function analysis of FrUDPG, and regulatory mechanisms of FrMYB1 and FrMYB2. Firstly, we will clone the full length of FrUDPG using RACE method, and then analyze the time and spatial expression profile of FrUDPG by real-time PCR. In addition, we also determine the flavonoid content and composition and reveal the correlation between gene expression and flavonoid content. Secondly, the combination of prokaryotic expression and transgenic plant determine the biofunction of FrUDPG. Finally, we investigate the mechanism of FrMYB1 and FrMYB2 in regulating the transcriptional expression of FrUDPG, and whether FrMYB1 and FrMYB2 could interact with other proteins. The present study will provide fundamental basis and study model for understanding flavonoid modification and regulatory mechanisms, and offer important gene resources for developing new plant cultivars with healthy function. Therefore, this research has the double meaning of theory and practice.

金柑是集药食两用和观赏于一身的我国特色资源植物，其果实中含有丰富的非花青苷类黄酮，是研究类黄酮合成调控的理想材料。基于转录组数据挖掘，我们从金柑中获得1个类黄酮糖基转移酶基因（FrUDPG）和两个潜在的上游转录因子（FrMYB1 和FrMYB2）。本项目围绕FrUDPG的生物功能解析及FrMYB1 和FrMYB2调控模式展开研究。利用Real-time PCR分析FrUDPG基因的表达模式，同时利用UPLC检测类黄酮含量，分析基因表达与类黄酮之间的相关性；随后，结合原核表达和转基因技术对FrUDPG生物功能进行解析。最后，分析FrMYB1和FrMYB2对FrUDPG基因转录水平的调控机理，并挖掘FrMYB1和FrMYB2是否存在互作蛋白。项目的开展深化了人们对植物非花青苷类黄酮合成调控机制的认识；另一方面也为开发具有特种保健功能的植物新品种提供了具有自主知识产权的基因资源。

金柑是我国的一种特色资源植物，果实中含有丰富的类黄酮。基于转录组数据分析，我们从金柑中获得1个类黄酮糖基转移酶基因（FrUDPG）和两个潜在的上游转录因子（FrMYB1和FrMYB2）。功能分析发现，FrUDPG具有Flavone O-glucosyltransferase, Flavonol O-glucosyltransferase, Flavonol O-rhamnosyltransferase酶活性。由于金柑中，含有糖基化的黄酮和黄烷酮，不包含糖基化的黄酮醇。因此，我们认为FrUDPG参与了黄酮（Flavones）氧糖基化修饰。进一步的分析发现，FrUDPG的表达和果实中糖基化黄酮呈现正相关。这些结果暗示，FrUDPG主要参与了金柑果实糖基化黄酮的修饰过程。随后，我们采用酵母单杂分析发现，两个转录因子和FrUDPG之间没有互作。但由于转录组分析过程中，这两个转录因子与类黄酮合成及修饰基因在表达量上紧密聚类，因此我们认为这两个转录因子下游靶基因可能是其他的类黄酮合成基因，而非FrUDPG。项目的开展深化了人们对植物非花青苷类黄酮合成调控机制的认识；另一方面也为开发具有特种保健功能的植物新品种提供了具有自主知识产权的基因资源。