牡丹花色形成相关MYB类转录因子的分离及其功能分析

Flower color is an important qualitative character and economic barometer for tree peony (Paeonia suffruticosa). The illumination of flower coloration regulatory mechanism provides a theoretical basis for flower color breeding. Anthocyanin accumulation is mainly controlled through the expression of structural and regulatory genes, and MYB transcription factor is the most important regulator factor involved in anthocyanin metabolic pathway. In our previous study, full-length cDNAs of six structural genes, including CHS, CHI and so on, were isolated from tree peony, and the expression characteristics of them were preliminarily analyzed using RT-PCR. In this study, petal tissue of different colored flowers will be collected at different opening stages as materials, and features of its transcriptome will be characterized at the gene level through analysis of RNA deep sequencing (RNA-Seq) data. In order to identify the MYB target gene involved in transcriptional regulation of tree peony flower coloration, MYB transcription factors will be predicted by bioinformatics, and then a comprehensive analysis on the expression patterns of predicted MYB and the six structural genes will be carried out using qRT-PCR. Subsequently, functional analysis of the target gene will be performed by overexpression and RNA interference (RNAi) in transformed tobacco. The results of this project will pave the way to elucidate the regulation mechanism of MYB and molecular basis in tree peony flower coloration and can also facilitate to the development of new cultivars of tree peony with different colors by manipulating flavonoid structural and regulatory genes through biotechnology techniques.

花色是牡丹重要的质量性状和经济指标，阐明其形成的调控机理是开展花色育种的理论基础。植物花色的形成主要受结构基因和调节基因的控制，其中MYB转录因子是花色素苷代谢途径中涉及最广泛的调节因子。课题组前期分离了牡丹CHS、CHI等6个结构基因cDNA全长，并通过RT-PCR初步分析了它们在不同色系品种花朵着色进程中的表达特性。在此基础上，本项目拟采用RNA-seq技术对不同颜色各开放阶段的花瓣进行转录组测序，经生物信息学手段预测MYB家族因子，通过qRT-PCR技术综合分析其与结构基因在不同花色形成过程中的表达模式，进而筛选出参与花色形成调控的目标MYB基因，然后利用转基因烟草超表达和RNAi抑制表达对目标基因进行功能分析。研究结果可望为阐明MYB对牡丹花色形成的调控机制奠定基础，并对揭示牡丹花色形成分子机理，加快我国牡丹花色分子育种进程具有重要的理论意义和实践价值。

牡丹是我国的传统名花，花色是牡丹重要的观赏性状和经济指标。植物花色的形成主要受结构基因和调节基因的控制，其中MYB转录因子是花色素苷代谢途径中涉及最广泛的调节因子。为探讨MYB转录因子对牡丹花色形成的分子调控机制，本研究采用RNA-seq技术对不同颜色各开放阶段的牡丹花瓣转录组进行测序和分析，并应用聚类分析和序列同源性比对等方法，从牡丹花瓣RNA-seq数据库中21个注释为R2R3MYB的Unigene中筛选到1个调控花色素苷生物合成的候选成员，命名为PsMYB1。利用qRT-PCR技术对PsMYB1和前期获得的牡丹类黄酮生物合成相关结构基因的表达模式进行分析，结果表明，PsMYB1的转录丰度在不同花色的花朵着色进程中及不同组织中均与花色素苷的生物合成呈密切相关，并推测其通过调控DFR、ANS和3GT等下游基因的表达参与牡丹类黄酮生物合成的转录调控。超表达PsMYB1转基因烟草花色明显加深，花色素苷含量和相关结构基因的表达水平均相应上升；而转化PsMYB1 RNAi抑制表达载体的烟草花色显著变浅，花色素苷含量和相关结构基因的表达水平也明显下降。此外，本研究还对转录组测序获得的12个牡丹内参基因的稳定性进行了评价，筛选出在牡丹不同花色、不同组织和不同开放阶段的最佳看家基因组合为UPL和PP2A。以上研究结果不仅为阐明MYB转录因子对牡丹花色形成的调控机制奠定基础，并对揭示牡丹花色形成分子机理，加快我国牡丹花色分子育种进程具有重要的理论意义和实践价值。