APETALA2基因启动子区甲基化修饰对托桂型芍药花型形成的调控机制分析

培育花型多样且具有较高观赏和商业价值的芍药新品种备受关注。然而，目前关于芍药花型形成相关的分子机制尚不清楚，遗传改良进展缓慢。申请人前期以花型独特雅致的托桂型芍药为研究对象，利用多组学测序结合功能验证等方法，已筛选鉴定出花型调控关键候选基因—APETALA2。本研究将锁定APETALA2启动子区甲基化修饰对托桂型芍药花型形成的调控作用，利用生物信息学结合实验手段，确定APETALA2核心启动子区；利用BSP-Miseq测序对其进行甲基化位点检测，筛选重要甲基化位点及转录调控因子，并利用ChIP和EMSA实验进行体外验证，同时，构建重要转录因子的过表达载体，从个体水平上验证转录调控元件对基因表达及芍药花瓣形态发育的影响。分析DNA甲基化修饰对基因表达及芍药花型形成的调控作用，从而阐明APETALA2基因表观遗传修饰对芍药花型调控的分子机制，为我国芍药花型分子育种的理论研究与育种实践提供参考。

The cultivation of Paeonia lactiflora with various flower types and high ornamental and commercial value has received much attention. However, the molecular mechanism of the forming of Paeonia lactiflora anemone petals remain unclear, so that genetic improvement has been slow. Based on the previous study that we selected Paeonia lactiflora with unique and elegant flower type as research object, and identified a key candidate gene regulating flower types-APETALA2. To further explored the regulation mechanism of APETALA2 promoter methylation on the forming of Paeonia lactiflora anemone petals, we identified the APETALA2 gene core promoter region by bioinformatics method combined with experimental verification, and detected methylation sites by BSP-Miseq sequencing. Through the analysis of the effect of methylation on gene expression, we screened out important methylation sites and transcription factors and further performed the validation experiment in vitro by ChIP and EMSA assays. Moreover, we conducted the overexpression of transcription factors, combining with the verification of genetic transformation system, we verified the effect of transcriptional regulatory elements on gene expression and flower types in Paeonia lactiflora. In this study, important methylation sites and transcriptional regulatory elements in APETALA2 core promoter region of Paeonia lactiflora were screened and verified. Thus, we systematically analyzed the effect of transcriptional regulatory elements on gene expression and flower types, in order to elucidate the molecular mechanism of DNA methylation modification regulating flower pattern in Paeonia lactiflora. Besides, this study provides theoretical reference for flower molecular breeding of Paeonia lactiflora in China.