GhAP1-D3基因调控陆地棉开花的分子机制解析

早熟性是作物提高复种指数，适应机械化采收的关键性状。开花时间是决定棉花早熟性最主要的性状之一。申请人前期利用QTL定位、GWAS、VIGS等方法，鉴定到一个调控棉花开花的关键基因GhAP1-D3，该基因启动子区DNA甲基化水平，在早晚花品种间存在显著差异；同时发现GhAP1-D3对环境温度反应敏感，适度高温能增强基因表达，并促使棉花提早开花。为深入探究GhAP1-D3调控陆地棉开花的分子机制，本项目拟利用过表达及基因编辑突变GhAP1-D3的转基因棉花材料，确定该基因在调控陆地棉开花中的作用；通过BSP、ChIP、酵母双杂等方法，明确DNA甲基化、组蛋白修饰水平与目的基因表达及开花时间的关系，并解析GhAP1-D3在棉花开花中的调控网络，阐明GhAP1-D3通过表观遗传修饰、响应环境温度变化参与调控陆地棉开花的分子机制。本研究将为棉花开花调控机制的揭示及优异早熟棉品种的选育奠定基础。

Early maturity is the important agronomic traits that contribute to the increase of the multiple cropping index and mechanization. Flowering time is one of the most important characters determining cotton early maturity. In our previous studies, a key gene GhAP1-D3 associated with early flowering was identified via quantitative trait loci (QTL) mapping, genome-wide association study (GWAS) and virus-induced gene silencing (VIGS). There were significant differences in DNA methylation in the gene promoter region between early- and late- flowering cotton varieties. Furthermore, we found upland cotton was sensitive to environmental temperature, and moderate high temperature can increase GhAP1-D3 gene expression and accelerate cotton flowering. To gain further insights into the molecular mechanism of GhAP1-D3 gene regulating flowering, the role of the gene regulating the flowering time will be elucidated in upland cotton, using transgenic cotton lines with over-expression and CRISPR/Cas9-mediated mutagenesis of GhAP1-D3. The relationship between the level of DNA methylation and histone modification and GhAP1-D3 gene expression and flowering time will be clarified, and the regulatory networks of GhAP1-D3 will be revealed in cotton flowering by several methods including bisulfite sequencing PCR (BSP), chromatin-immunoprecipitation (ChIP), and yeast double hybridization in the study. Importantly, we will investigate the molecular mechanism of GhAP1-D3 regulating upland cotton flowering through epigenetic modification in response to environmental temperature changes. This research will lay the foundation for uncovering the regulating mechanism of cotton flowering and improving the early maturity cotton varieties.