牡丹花瓣红色和紫色形成的分子机制

Tree peony (Paeonia section Moutan DC.) is one of the top ten traditional flower plants in China. It possesses multi-functional values including ornamental, medicinal, oil-utilized, and other economic values. Tree peony confers various flower colors, such as red, pink, purple, yellow, and green, which is an excellent material for the studies on mechanism of flower color formation and identification of the related functional genes. After 20 years of endeavors, the applicants have analyzed and identified 38 pigments in petals of tree peony, which are all flavonoids. We also characterized the functions of the flavonoid O-methyltransferase gene (PsAOMT) and idenitified that it was one of the key genes accounting for purplish flower coloration of tree peony. However, although the chemical mechanism has been basically clear, the molecular meachnism of different coloration has yet to be discovered. Therefore, this project is going to study typical red- and purple-colored cultivars, to analyze the composition of flavonoids in petals, and further to discover the relationship between pigment constituents and color formation. Secondly, we will put emphasis on the characterization of the key structural genes (F3’H, 3GT, and 5GT) as well as regulatory genes (MYB, bHLH, and WDR) in petals of red- and purple-colored flowers of tree peony, in order to elucidate the roles of glycosylation and methylation of flavonoids in flower coloration. This study will provide us with a thorough understanding on the underlying molecular mechanism of color formation of tree peony flowers, which will be beneficial for molecular breeding as well as germplasm innovation of flower plants.

牡丹是我国十大传统名花之一，具有观赏、药用和油用多种经济价值。牡丹被誉为“国色天香”，具有红、粉、紫、黄、绿、白等丰富的花色变异，是阐明“国色”形成机制、挖掘花色功能基因的宝贵资源。申请人及其研究组经过20余年的努力，分析并鉴定了牡丹花瓣中38种色素，它们都属于类黄酮化合物；我们通过牡丹类黄酮甲基转移酶基因（PsAOMT）功能研究，证实了该基因是导致牡丹紫色化的关键基因之一。迄今为止，虽然牡丹花色形成的化学机制已基本清楚，但其不同色系形成的分子机制尚待逐一解明。本项目拟以分布频度高的红、紫色系典型品种为材料，分析其花瓣的类黄酮组成，探讨色素组成与花色的关系；其次，克隆与牡丹红、紫色相关的关键结构基因（F3’H、3GT、5GT）和调控基因（MYB、bHLH和WDR），并进行功能验证，重点阐明类黄酮糖苷化、甲基化与花色的关系，深入解析牡丹红色和紫色形成的分子机制，为牡丹花色育种奠定理论基础。

牡丹是我国十大传统名花之一，具有红、粉、紫、黄、绿、白等丰富的花色变异。本项目以分布频度高的红、粉、紫色系典型品种（分别为‘太阳’、‘胡红’、‘岛大臣’）为材料，分析了其花瓣的类黄酮组成，探讨了色素组成与花色的关系，发现到盛开期时，紫色花中的花青素苷总量分别为红色、粉色花瓣中的2倍和9倍。紫色花中的花青素苷种类最多，红色花次之，粉色花最少。紫色花中主要含有芍药花素 3,5-二葡萄糖苷（Pn3G5G）；粉色和红色花中分别以天竺葵素 3,5-二葡萄糖苷（Pg3G5G）和天竺葵素 3-葡萄糖苷（Pg3G）为主；三者的黄酮苷、黄酮醇苷总含量相近，三个品种的花瓣中都含有高含量的芹菜素7-新橙皮糖苷（Ap7N），而红色花瓣中山奈酚3,7-二葡萄糖苷（Km3G7G）的含量明显高于紫色花和粉色花。从转录组数据中共检索到类黄酮合成相关酶基因243个、调控基因7个；利用实时荧光定量结果确定了候选酶基因和候选调控基因，共克隆得到了34个酶基因和4个调控基因。构建了原核表达载体，进行了类黄酮甲基转移酶（PsOMT）和类黄酮糖基转移酶（Ps3/5GT）的体外酶活功能验证，已获得4个具有活性的PsMT、3个PsF5GT和2个PsF3GT。克隆得到了一个具有体外活性的类黄酮甲基转移酶基因（PsMT4）的启动子，并分析了该启动子和4个候选PsMYB转录因子的序列。已得到可用于农杆菌侵染的牡丹愈伤；利用基因枪轰击法建立了牡丹花瓣基因功能的体内验证技术体系。本项目重点阐明了牡丹类黄酮甲基化、糖基化与花色的关系，深入解析了牡丹红色和紫色形成的分子机制。