芍药花瓣黄色变异的分子调控机制研究

Yellow is the rarest flower color in herbaceous peony (Paeonia lactiflora Pall.), but it is very difficult to breed new yellow germplasm according to conventional breeding methods. Therefore, petal yellow mutation in herbaceous peony has great commercial development value and research value. In this project, the flavonoid metabolic pathway directly related to the development of flower color will be used as the main line, and herbaceous peony chimera including outer red petals and inner yellow mutations will be used as the test material to determine the changes of flavonoids, pH value, organic acid and metal ion contents, analyze related genes expression levels in the flavonoid metabolic pathway, find key structural genes controlling the formation of yellow, identify key cis-acting elements of key structural genes promoter regions and transcription factors through the analysis of gel retardation and yeast one-hybrid, and reveal the regulation mechanism of above-mentioned pathway participating in yellow character formation. Meanwhile, we will screen differentially expressed genes from transcriptomic sequencing, make clear a series of key genes participating in yellow character formation combining with gene function verification, and construct related metabolic pathway controlling petals yellow character formation in herbaceous peony. Finally, the above two research lines are contacted to validate and complement each other, and clarify molecular mechanism of petals yellow character formation from metabolic group, transcription group, regulation of promoter and transcription factor, which will provide a theoretical basis for genetic improvement of flower color in herbaceous peony.

黄色是芍药中最珍稀的花色，通过常规育种方法很难育出新的黄色种质，因此，芍药花瓣黄色突变具有重要的商业开发价值和研究价值。本项目以芍药同一花朵中外瓣红色、内瓣发生黄色突变的嵌合体为试材，以与芍药花色发育直接相关的类黄酮代谢途径研究为主线，测定类黄酮、pH值、有机酸及金属离子含量的变化，分析类黄酮代谢途径中相关结构基因表达，找到该途径中调控黄色形成的关键结构基因，通过凝胶阻滞和酵母单杂交分析鉴定关键结构基因启动子区域的关键顺式元件和转录因子，揭示上述途径参与黄色性状形成的调控机制；与此同时，再从转录组测序研究入手，筛选差异表达基因，结合基因功能验证，明确参与黄色性状形成的一系列关键基因，构建调控芍药花瓣黄色性状形成的相关代谢途径。最终，将上述两条研究线路联系起来相互验证补充，从代谢组、转录组、启动子与转录因子调控等多个层面阐明花瓣黄色性状形成的分子机制，为芍药花色性状的遗传改良提供理论依据。

黄色是芍药中最珍稀的花色，通过常规育种方法很难育出新的黄色种质，因此，芍药花瓣黄色突变具有重要的商业开发价值和研究价值。本项目以芍药同一花朵中外瓣红色、内瓣发生黄色突变的嵌合体‘金辉’为试材，以与芍药花色发育直接相关的类黄酮代谢途径研究为主线，通过测定类黄酮、pH值、有机酸及金属离子含量的变化发现，花黄素与花色苷之间存在的共色作用是芍药花瓣呈现黄色的主要原因，而与pH值、有机酸及金属离子含量关系不大。随后通过转录组测序，共获得61408条All-unigene，筛选到6345个差异表达基因（DEGs），它们主要富集于121条代谢途径上，其中有5条与色泽形成相关的代谢途径，分别是苯丙烷类生物合成、类黄酮生物合成、黄酮和黄酮醇生物合成、异黄酮生物合成和花色苷生物合成，它们均属于类黄酮代谢途径，并且以类黄酮生物合成途径为纽带构建了调控芍药花瓣黄色性状形成的相关代谢途径。在此基础之上，通过qRT-PCR技术初步筛选出影响芍药花瓣黄色性状形成的基因主要为PlPAL、PlFLS、PlDFR、PlANS、PlUF3GT和PlUF5GT。通过对这些基因全长序列的克隆、过量表达载体的构建以及对拟南芥等模式植物进行遗传转化，明确了PlDFR和PlANS在芍药花瓣色泽形成中的重要作用，从而最终阐明了芍药花瓣黄色性状形成的分子机制，为芍药花色性状的遗传改良提供理论依据。