miRNA介导芍药花瓣黄色性状形成的转录后调控机制研究

Yellow is the best color embodying “riches and honour" in flower culture of herbaceous peony (Paeonia lactiflora Pall.) and has a extremely high ornamental value, but there is scarce yellow P. lactiflora germplasm which is difficult to cultivate through conventional breeding methods, therefore understanding the molecular mechanism of the yellow character formation in P. lactiflora petals would facilitate the cultivation of new yellow P. lactiflora using genetic engineering. Our project team has initially identified the metabolic and transcriptional regulatory mechanisms of P. lactiflora yellow formation by previous studies, but its miRNA-mediated post-transcriptional regulatory mechanism is unclear. Based on the previous works, a color chimera with red outer-petal and yellow inner-petal is used as the material in this project to screen differentially expressed miRNA by miRNA sequencing, identify candidate miRNA controlling the formation of yellow by predicting target genes and constructing the regulatory networks of differentially expressed miRNA and its corresponding target genes, obtain key miRNA controlling the formation of yellow by gene functional verification, reveal the expressional and regulatory mechanisms of obtained key miRNA by key miRNA isolation and its promoter expression patterns, and then clarify miRNA-mediated post-transcriptional regulatory mechanism of the yellow character formation of P. lactiflora petals. Eventually, this result combined with previous studies would systematically elucidate the regulatory mechanism of the yellow character formation of P. lactiflora petals from multiple levels of metabolic regulation, transcriptional regulation, post-transcriptional regulation.

黄色是花文化中最能体现芍药寓意“富贵”的色彩，观赏价值极高，但黄色芍药种质稀缺并且很难通过传统育种方法培育，揭示芍药黄色形成的分子机理有助于运用基因工程培育黄色芍药新品种。项目组通过相关研究初步明确了芍药黄色形成的代谢调控和转录调控机制，但对miRNA介导的转录后调控机制尚不清楚。本项目拟在前期工作基础上，以外瓣红色、内瓣黄色的芍药花色嵌合体为试材，通过miRNA测序筛选黄、红色差异表达miRNA；根据预测的靶基因、构建的差异表达miRNA与靶基因之间调控网络来鉴定调控花瓣黄色性状形成的候选miRNA；结合基因功能验证，明确关键miRNA；接着开展关键miRNA启动子的分离与表达模式研究，揭示关键miRNA的表达调控机制，从而阐明miRNA介导芍药花瓣黄色性状形成的转录后调控机制。最终，与前期研究结果联系起来，从代谢调控、转录调控、转录后调控等多个层面系统地阐明花瓣黄色性状形成的调控机制。

黄色是花文化中最能体现芍药寓意“富贵”的色彩，观赏价值极高，但黄色芍药种质稀缺并且很难通过传统育种方法培育，揭示芍药黄色形成的分子机理有助于运用基因工程培育黄色芍药新品种。项目组在初步明确了芍药黄色形成的代谢调控和转录调控机制基础之上，以芍药外瓣红色、内瓣黄色的‘金辉’为试材，通过miRNA测序在红色外瓣和黄色内瓣中分别获得了1756525和2045228条miRNA，并且miRNA前体序列的亚克隆及表达模式的随机检测均验证了miRNA测序结果的准确性。通过差异分析，共筛选到在红色外瓣和黄色内瓣中差异表达的163个已知miRNA和28个新预测miRNA；根据它们对应靶基因的功能，共筛选到miR156e-3p、miR828a、miR156d、miR2616和Novel-miR25等5个调控芍药花色形成的候选miRNA。候选miRNA及其靶基因在不同发育时期芍药红色外瓣和黄色内瓣中的表达模式明晰了仅有miR156e-3p及其靶基因SPL1、miR828及其靶基因MYB114表达趋势呈显著负相关性，通过随后的拟南芥转基因研究肯定了miR156e-3p在调控色泽形成中的重要作用，它通过调控靶基因SPL1进而影响结构基因DFR的表达来调控植株色泽形成。这一结果阐明了miRNA介导芍药花瓣黄色性状形成的转录后调控机制，为运用基因工程技术培育黄色芍药新品种奠定了基础。