白菜花粉特异表达的BcWRKY31转录因子的功能及其靶标基因鉴定

Pollen development is one of the most important steps during the life cycle of flowering plants, with implications in hybrid production, yields and quality of seeds in vegetable crops. Our previous study has obtained a pollen specific WRKY transcription factor, BcWRKY31, from genechip analysis and bioinformation analysis in Chinese cabbage-pak-choi (Brassica campestris ssp. chinensis). In this study, we will use promoter fusing, quantitative real time-PCR and immunohistochemical stain analyses to determine the precise locations of the promoter, mRNA and their proteins. Then over-expression and RNAi technologies will be used to characterize the gene functions during pollen development. Morphological, cytological, palynological, physiological and biochemistrical differences of the transgenic plants will be monitored to examine the biological function of BcWRKY31 transcription factor. The pull down and the electrophoretic mobility shift assay will be performed to identify the target genes of the BcWRKY31. In addition, various environmental stresses including high temperatures, low temperatures, droughts and high humidity, will be applied to characterize the potential functions of BcWRKY31 during pollen development process. The morphological, and physiological and biochemistrical reaction of the plants will be observed, and the expression patterns of the BcWRKY31, its target genes as well the other 13 potential pollen WRKY genes will be identified. This study will reveal the working mechanism of WRKY transcription factors during pollen development as well as their responses to stresses. Results shall help to better understand the mechanism of male sterility, and the control of fertility in plants.

花粉发育是种子植物完成生活周期的重要环节，直接关系到蔬菜等农作物杂种一代的生产、种子与果实的产量高低等一系列农业问题。项目在前期基因芯片分析获得白菜花粉特异表达的BcWRKY31转录因子和通过生物信息学分析获得另外13个可能在白菜花粉中高或特异表达的WRKY基因的基础上，通过启动子融合表达、qRT-PCR、免疫组化分析明确BcWRKY31的确切表达位置；构建过表达和RNAi载体进行遗传转化，分析转化植株的形态学、细胞学、孢粉学、生理生化等性状差异；采用pull down技术和凝胶迁移技术分析BcWRKY31的靶标标基因；另外，对上述转基因植株进行高温、低温、干旱及高湿等胁迫处理，分析BcWRKY31、其靶标基因，及其他13个可能的白菜WRKY基因在花粉胁迫响应中的功能。这些研究对于深入认识WRKY转录因子在花粉发育过程中的作用，有效控制植物育性等有着十分重要的理论意义与应用价值。

花粉发育是种子植物完成生活周期的重要环节，直接关系到蔬菜等农作物杂种一代的生产、种子与果实的产量高低、质量好坏和品质优劣等一系列农业问题。项目在前期基因芯片分析获得白菜花粉特异表达的BcWRKY31转录因子和通过生物信息学数据库分析获得另外13个可能在白菜花粉中高或特异表达的WRKY基因的基础上，通过qRT-PCR等方式明确了BcWRKY31的确切表达位置，明确了BcWRKY31的确切表达部位为四级花蕾，并在授粉后10表达量提高9倍；其次确定了白菜WRKY基因家族成员有至少有137个，并对他们的进化模式进行了分析；构建过表达和RNAi载体进行遗传转化；构建了WRKY的融合表达蛋白用以分析BcWRKY31、其靶标基因，及其他13个白菜中在花粉中高或特异表达的WRKY基因在花粉胁迫响应中的功能。项目还对WRKY在生物胁迫、非生物胁迫，以及其WRKY一起下游基因以及WRKY家族基因之间的调节作用进行了细致的分析，为更好解析WRKY31的作用模式做了很好的专题分析。这些研究对于深入认识WRKY转录因子在花粉发育过程中的作用，有效控制植物育性或结实性等有着十分重要的理论意义与应用价值。