白菜CRFs基因在雌蕊发育中的作用及分子调控机制研究

Result indicates that the precise maintaining and termination of floral meristem is the critical point for gynoecia tissues differentiation and morphogenesis of gynoecium in plant. Gynoecium development plays an important role in ontogenesis and alternation of generations in higher plant, and has an important effect on crop yield and quality. In this study, the temporal and spatial expression characteristics of CRFs gene and their response to different CK will be studied by qPCR and in situ hybridization (ISH) in Chinese cabbage (cv. Bajh97-01B). Subsequently, the artificial microRNA (amiRNA) method will be employed to analyze the biological functions of BrCRF1 and BrCRF19 from Chinese cabbage, and whether there is functional redundancy will be illuminated. Meanwhile, Yeast two-hybrid, BiFC, GST pull-down and ChIP will be performed to study the regulation and crosstalk between CRFs and transcriptional factors which involved in phytohormone signaling pathway. The function of CRFs will be clarified on the process of the precise maintaining and termination of floral meristem. At last, we aim to analyze the relationship which mediated by CRFs between the miRNA and epigenetic patterns and the termination of floral meristem. Results will be helpful to elucidate the molecular mechanism of CRFs regulation of the pistil development through the transcription feedback and epigenetic regulation in Chinese cabbage. In conclusion, these results of this study will provide insight into the molecular mechanism of sexual reproduction in economic crops. In addition, it will also supply the novel theoretical basics to regulate the fertility and realize the high yield and good quality in seed production in Brassica crops.

花分生组织精准的维持和终止是植物雌蕊组织分化和形态建成的关键环节，雌蕊发育在高等植物个体发育和世代交替过程中占有重要地位，对农作物的产量和品质有着根本性的影响。目前我们对CRFs在植物雌蕊发育中的作用及分子机理还知之甚少。本项目拟以白菜‘Bajh97-01B’为材料，在分析BrCRFs的时空表达特征及其对不同细胞分裂素的响应特征的基础上，应用amiRNA方法分析BrCRF1和BrCRF19的生物学功能。同时，采用酵母双杂与BiFC以及GST pull-down与ChIP-seq等技术研究BrCRFs与植物激素信号传导途径相关转录因子间的表达调控关系，阐明BrCRFs在花分生组织的维持和终止过程中的作用。随后，分析miRNA和组蛋白甲基化与BrCRFs间的作用关系，揭示BrCRFs通过转录反馈与表观遗传等途径来调控白菜雌蕊发育的分子机制。研究结果将为实现蔬菜的高效繁育提供研究依据。

花分生组织精准的维持和终止是植物雌蕊组织分化和形态建成的关键环节，雌蕊发育在高等植物个体发育和世代交替过程中占有重要地位，对农作物的产量和品质有着根本性的影响。目前我们对CRFs在植物雌蕊发育中的作用及分子机理还知之甚少。本项目以白菜‘Bajh97-01B’为材料，在开展U三角作物CRFs基因家族分析的基础上，共鉴定出141个CRF基因家族成员，根据其C端序列相似性可以分为5个进化枝，对其蛋白结构的比较发现存在3种结构模型。随后，分析BrCRFs的时空表达特征及其对不同细胞分裂素的响应特征，应用amiRNA方法分析BrCRF6和BrCRF11a的生物学功能。观察发现，改变BrCRF6和BrCRF11a的表达会导致白菜花发育异常，产生了花中有花的嵌套花现象。同时，采用酵母双杂与BiFC以及pull-down与CoIP等技术研究BrCRFs与植物激素信号传导途径相关转录因子间的表达调控关系，发现BrCRF6和BrCRF11a蛋白可以与BrWUSs蛋白以及细胞分裂素信号转导相关蛋白BrHPs以及B类BrRRs相互作用，并且CRF结构域在互作过程中发挥关键作用。BrCRF6和BrCRF11a可以与花分生组织发育相关蛋白互作影响雌蕊的发育，并且BrCRF6和BrCRF11a参与了植物激素调控途径。结果说明，BrCRFs在花分生组织的维持和终止过程中起重要作用。基因功能分析结果显示，BrWUS1在植物营养生长和生殖发育过程中均具有重要作用，但BrRR10和BrRR12主要在植物营养生长阶段发挥作用。最后，我们正在分析miRNA和组蛋白甲基化与BrCRFs间的作用关系，揭示BrCRFs通过转录反馈与表观遗传等途径来调控白菜雌蕊发育的分子机制。研究结果将为实现芸薹属作物的高效繁育提供理论依据和技术支撑。