白菜花粉发育相关MYB转录因子受miR319调控通路的研究

本项目在发现白菜花粉发育相关MYB转录因子编码基因BcMYB81/104及其上游调控因子bra-miR319的基础上，拟利用CRISPR-Cas9基因敲除和转基因等技术，结合亚细胞定位和时空表达模式分析，研究BcMYB81/104的功能；采用过表达、GFP瞬时表达及GUS融合表达体系，研究bra-miR319调控BcMYB81/104的机制，并结合已有研究，比较miR319与miR159对MYB调控的异同；通过染色质免疫共沉淀测序和凝胶电泳迁移实验筛选并验证BcMYB81/104下游靶基因，并对下游靶基因进行功能分析，从而构建“microRNA–MYB转录因子–下游靶基因”在花粉发育过程中的调控网络。这些研究将为有效控制蔬菜等农作物育性提供理论依据。

On the basis of discovering a MYB transcription factor BcMYB81/104, which is related to pollen development in Brassica campestris ssp. chinensis, and its potential upstream regulator bra-miR319, we plan to analyze the temporal and spatial expression patterns of BcMYB81/104, and then use the CRISPR-Cas9 and transgenic technology to analyze its biological functions during the processes of pollen development. Over-expression, GFP and GUS fusion expression systems will be used to unveil the regulation of BcMYB81/104 by bra-miR319. Combining with the research findings existed previously, the similarities and differences between the regulations of MYB by miR319 and/or miR159 will be analyzed. Meanwhile, ChIP and EMSA assays will be chosen to screen and verify the putative downstream target genes of BcMYB81/104, then more researches will be conducted to analyze the function of these genes. By building the regulatory network of “microRNA-MYB-MYB target genes”, we can gain further understanding of the complicated regulations of MYB in pollen development from the perspective of interactions between microRNA and MYB, MYB and its target genes. What’s more，these results will lay a considerable theoretical foundation for the effective fertility control in important crops especially in vegetable crops.