采用转录组及代谢组关联解析高山杜鹃典型花色形成的分子机制

Alpine rhododendrons are world famous woody ornamental plants, which were discovered in China and thrived abroad. A large number of Chinese specific alpine Rhododendron species, with great potential for ornamental values, need to be studied and explored. This projectis based on the study results of flower colors and pigments of more than 30 Rhododendron species. Two alpine rhododendrons with typical flower colors (purple-flowered Rhododendron davidii and red-flowered R. delavayi) are selected. According to the flavonoids constitutions and contents in two species, it is speculated that the flavonoid metabolic pathways in two species have undergone different regulations, resulted in the differentiation of expression levels or protein activities of several key structural genes, which leads to the variation between flavonoids compositions and flower colorations. This project is going to analyze the transcriptomes during flower development using RNA sequencing, and using bioinformatics methods to analyze and compare the differences in the regulation and structural genes related to flavonoid biosynthesis pathway, and verified their expression variations during development by real-time PCR analyses. Integrated with the pigment composition and accumulation measured by liquid chromatography, to perform associated analysis between transcriptome and flavonoid metabolism data, in order to identify the difference in regulatory and structural genes, then further investigate the sequence polymorphism, gene function, and cis-elements regulation of different genes. By combination of the results, the molecular mechanism of color formation and variation of the two typical flower colors in alpine rhododendrons will be elucidated. The project will shed light on molecular breeding and cultivars innovation of alpine rhododendrons.

高山杜鹃是源自我国的世界著名木本花卉，我国特有大量极具观赏价值的高山杜鹃种质资源，尚待系统研究和开发利用。本项目根据对30余种杜鹃花色及类黄酮色素组成研究结果，选择具有典型紫色、红色花的腺果、马缨杜鹃为研究对象，根据已获得的类黄酮色素组成信息，推测其类黄酮代谢途径经历了不同的调控作用，导致合成途径上关键结构基因表达或功能的差异，进而导致类黄酮色素组成和色彩形成的变异。本项目拟采用转录组测序分析不同发育阶段花瓣的转录本，利用生物信息学手段比较分析类黄酮代谢调控和结构基因的差异，并采用荧光定量PCR在不同发育阶段花瓣中进行表达量验证，结合液相色谱测定类黄酮色素种类和积累过程，关联分析转录组与类黄酮代谢数据，寻找差异的调控基因及其调控的关键结构基因，并从基因多态性、功能变异、顺式作用元件等方面研究其调控机制，解析高山杜鹃典型花色变异的分子机制，为高山杜鹃新品种培育和开发利用提供理论依据。

杜鹃是世界著名木本花卉，我国拥有大量特有的高山杜鹃种质资源，然而研究和开发利用尚处于起步阶段。因此，本项目以具有典型紫色、红色花的高山杜鹃为研究对象，采用液相色谱-质谱联用技术，测定不同发育阶段的花瓣中类黄酮色素等次生代谢产物的组成和积累情况。共定性定量了花青苷5种、绿原酸及异构体3种、及黄酮醇14种。结果显示两种杜鹃花色素组成和含量上均有差异，马缨杜鹃以a4和a5矢车菊素糖苷为主，而腺果杜鹃a1和a3飞燕草素糖苷为主，且花青苷总含量前者为后者5倍多。同时，利用转录组测序分析比较不同发育阶段花瓣的转录本，挖掘类黄酮代谢结构基因的差异和相关的调控因子。共拼接得到172,071个转录本，99471个Unigene，多重比较发现多个差异表达基因可能在类黄酮合成途径中起重要作用。并采用荧光定量PCR在不同发育阶段花瓣中进行表达量验证。以代谢组数据作为表型数据与转录组结果进行加权基因网络共表达分析（WGCNA），共得到38个模块，其中orangered3模块与a1 - a5的Pearson相关系数绝对值最高，分别为0.99、 0.98、0.99、0.94和0.95，表明orangered3可能是与花青苷合成和积累最相关的模块。其中，类黄酮半乳糖苷转移酶基因C3L33_16771与花青苷积累相关性很高，因此本项目提出由于糖基转移酶大量表达，导致花青苷积累速度快、积累量大，导致杜鹃花深红的花色形成的假设，并挖掘出一批有深入研究价值的转录调控因子，为高山杜鹃新品种培育和开发利用提供理论依据。