Bol-miR028介导的F-box表达对甘蓝雄性不育的调控机制研究

MicroRNA (miRNA) plays an important role in the growth and development of plants by mediating the degradation or translation of target mRNA to inhibit the expression of regulatory genes in the post-transcriptional level. In this study, we have screened and obtained a pollen- specific small RNA Bol-miR028 and its targeting gene BoFbx1 encoding F-box protein. On the basis, their expressional character is analyzed by the promoter-GUS fusion expression and green fluorescent protein (GFP) localization. The biological functions of Bol-miR028 targeting the regulation of BoFbx1 is clarified during the pollen development using over-expression technology and site-directed mutagenesis. Then, their functional downstream genes are screened by yeast-two hybrid technique. The expression levels of Bol-miR028, BoFbx1 and their downstream genes and proteins are identified and analyzed in all transgenic plants and mutants by qRT-PCR and Western blot, so as to explore the regulation mechanism of Bol-miR028 mediated F-box protein silencing in gene expression networks on male sterility. This study not only lays the foundation for the accurate interpretation of the molecular mechanism of miRNA regulating the plant pollen development, but also has an important guiding significance for the rapid establishment of crop male sterile line by using artificial miRNA technology.

MicroRNA（miRNA）在转录后水平通过介导靶mRNA的降解或翻译抑制调控基因表达，在植物生长发育中发挥重要作用。项目组前期筛选获得一个新的甘蓝花粉特异小RNA分子Bol-miR028，其靶基因BoFbx1编码F-box蛋白。本项目拟在此基础上通过启动子-GUS融合表达、绿色荧光蛋白（GFP）定位等方法明确目标基因的表达特征；采用过表达和基因定点突变技术，研究Bol-miR028靶向调控BoFbx1在花粉发育中的生物学功能；通过酵母双杂交系统筛选下游基因，并运用qRT-PCR和Western blot鉴定转基因植株中目标基因及其下游基因和蛋白的表达水平；从而探究Bol-miR028介导的F-box沉默涉及的基因表达网络对甘蓝雄性不育的调控机制。本研究不仅为精确解读miRNA调控植物花粉发育的分子机制奠定基础，而且对利用人工miRNA技术快速创建雄性不育系具有积极的指导意义。

植物中存在许多miRNA分子，它们在植物的生长发育、激素信号转导和胁迫应答反应中发挥着重要的功能。甘蓝花粉发育相关miRNA的发现及其调控机制的研究对于深入了解植物的生殖规律和雄性不育的机理，具有重要意义。项目研究了甘蓝bol-miR028及其靶基因BoFBX156在花粉发育中的生物学功能；揭示了bol-miR028与BoFBX156靶向关系及调控作用机理，明确了甘蓝F-box基因家族中关键花粉调控和低温相关基因，从而探究bol-miR028介导的F-box蛋白沉默对甘蓝花粉发育的调控机制，为揭示miRNA在植物花粉发育中的分子机理奠定重要的理论基础。同时获得了具有自主知识产权的，功能显著的甘蓝miRNA和F-box新基因及转基因甘蓝新材料，为培育创制甘蓝新品种提供了宝贵的基因资源和植物材料。