表观调控因子BrVIN3控制大白菜抽薹时间的分子机制研究

Early bolting is one of the major problems responsible for restricted production of Chinese cabbage in spring and highland cultivation. The primary solution is exploring bolting-resistant gene and breeding late-bolting varieties. Therefore, clarifying the molecular mechanism of bolting time control is significantly important to improve the breeding efficiency of Chinese cabbage. In our previous study, a QTL for flowering time was detected on A02 chromosome based on SLAF sequencing and genome-wide association analysis, which was consistent with the QTL determined by traditional genetic mapping. Moreover, with representative germplasm sequencing and gene expression analysis, an epigenetic regulation factor, BrVIN3, which locate in the QTL interval was proposed to be the candidate gene that involved in bolting time control. In this proposal, we will further verify the function of BrVIN3 by candidate gene association analysis. The expression pattern analysis of BrVIN3 during vernalization, transgenic plants analysis and cytological study will be conducted to illuminate the gene function. Chromatin Immunoprecipitation (ChIP), Pull-Down and Yeast-2-Hybrid experiments will also be performed to explore which BrFLC (BrFLCs) is (are) epigenetically repressed by BrVIN3 and what proteins are involved in the target specificity determination of BrVIN3. The study of BrVIN3 will explore the underlying molecular mechanism of bolting time control and develop functional molecular markers of BrVIN3, which will lay a foundation for large-scale screening of bolting resistant resources and molecular assisted selection of late bolting trait in Chinese cabbage breeding.

培育抗抽薹新品种是解决我国春夏大白菜先期抽薹开花问题的根本途径，阐明大白菜先期抽薹的分子机制，是提高育种效率的必由之路。基于SLAF测序和全基因组关联分析，结合QTL遗传定位，我们在白菜A02染色体定位了一个控制低温需求型白菜抽薹开花的QTL，利用代表性白菜种质重测序和表达分析证实该区间的表观调控因子BrVIN3是控制白菜抽薹开花的关键候选基因。本项目拟采用候选基因关联分析方法证实该基因是控制白菜抽薹开花的关键基因，并从转基因、表达模式和细胞学水平对BrVIN3进行功能解析，阐述BrVIN3在调控白菜抽薹过程中的作用，开发BrVIN3基因的分子标记；利用染色质免疫沉淀、Pull-Down和酵母双杂交等手段获得BrVIN3特异作用的靶标BrFLC和BrVIN3互作蛋白因子，初步构建BrVIN3控制白菜抽薹时间的分子调控网络。该研究将为深入探索白菜晚抽薹的分子机理和晚抽薹分子辅助育种奠定基础。

利用SLAF测序和全基因组关联分析，结合QTL遗传定位，在白菜A02染色体定位了一个控制低温需求型白菜抽薹开花的关键基因BrVIN3-1。利用拟南芥和白菜转基因证实BrVIN3-1正向调控植物开花，BrVIN3-1的表达模式和细胞定位分析进一步证实了BrVIN3-1的功能。进而单基因表型关联和基因单体型分析证明BrVIN3启动子差异是导致白菜抽薹差异的关键因素。启动子-GUS活性分析显示不同材料中的BrVIN3-1启动子在春化处理后驱动基因表达的能力显著不同。利用启动子序列差异开发的功能型SNP标记已经应用于分子标记辅助选择育种。最后利用染色质免疫沉淀 （Chromatin Immunoprecipitation assay， CHIP）和Pull-Down等研究手段获得了BrVIN3特异作用的靶标BrFLC和BrVIN3-1互作蛋白因子，初步构建了BrVIN3-1控制白菜抽薹时间的分子调控网络。该研究为深入探索白菜晚抽薹的分子机理和晚抽薹分子辅助育种奠定了基础。