甘蓝雄性不育相关miRNA的分离鉴定与表达调控研究

MicroRNA(miRNA) is significant in the regulation of plant growth and development, but there is no systematic study on the miRNA related to pollen development in cabbage (Brassica oleracea var. capitata L.). In this study, we have utilised large-scale deep sequencing to explore the small RNA library in flower bud of cabbage. On the basis, candidate miRNAs involved in cabbage pollen development were predicted by means of bioinformatics methods. We will screen miRNAs related to male sterility by comparing the expression profiles of pollen-specific miRNAs in the cabbages with different fertility using the miRNA chip, and test them by combining Northern blot hybridization and special stem-loop RT-PCR technique. Then, we will predict and verify target genes of the new miRNAs, analyze their function, and clone the target genes closely associated with male sterility. In addition, using qRT-PCR and in-situ hybridization method, we will study the temporal and spatial expression patterns of the key miRNAs and target genes in pollen development, in order to clarify the gene expression regulation effect of miRNA on the male sterility in cabbage. The project would provide novel insights into understanding the systematic regulation of pollen development and elucidating the molecular mechanism of male sterility. Furthermore, it would lay a foundation for finally regulating the pollen fertility of Brassica crops and promoting the extensive use of heterosis by artificial miRNA technology.

MicroRNA（miRNA）在植物生长发育中具有重要调控作用，但甘蓝花粉发育相关miRNA尚未见系统研究。本项目在对甘蓝花蕾小RNA文库进行大规模深度测序的基础上，结合生物信息学技术获取甘蓝花粉发育候选miRNA；并利用miRNA芯片比较不同育性甘蓝花粉特异miRNA的表达谱变化，筛选雄性不育相关miRNA，通过Northern杂交和特殊的茎环RT-PCR技术对其检测鉴定；预测新的miRNA的靶基因并进行功能分析和实验验证，克隆与雄性不育密切相关的靶基因；采用实时荧光定量PCR和组织原位杂交方法，研究关键miRNA和靶基因在不同育性甘蓝花粉发育过程中的时空表达特征，从而明确miRNA对甘蓝雄性不育的基因表达调控作用。项目的执行将为全面认识花粉发育的系统性调控，揭示植物雄性不育的分子机制提供新的理论依据，并为最终通过人工miRNA技术调控芸薹属作物花粉育性，推动杂种优势的广泛利用奠定基础。

本项目通过对甘蓝花蕾小RNA文库进行大规模深度测序，结合生物信息学技术获取大批甘蓝花粉发育候选miRNA及其靶基因。通过茎环RT-PCR技术对雄性不育相关的bol-miR157a，bol-miR171a，bol-miR172，bol-miR398a-3p，bol-miR824和其他关键miRNAs进行实验验证和分析鉴定。通过实时荧光定量PCR研究关键miRNAs和靶基因在不同育性甘蓝花粉发育过程中的时空表达特征，发现miRNAs在甘蓝花粉发育的各个时期均有表达且表达量差异较为明显，并且在保持系中的单核小孢子期表达量最高而在雄性不育系的双核花粉期表达量最高，在花粉成熟期和花粉母细胞时期表达量最低。而且miRNAs的靶基因大多为调节基因表达的转录因子以及信号转导途径中相关的酶等，这些靶基因大多参与调节植物器官形态建成和植物花粉发育过程。对甘蓝miRNA靶基因SET结构域基因和DHHC型锌指蛋白基因进行家族鉴定与表达特征分析，表明其在甘蓝花粉发育过程中表达量存在显著差异，推测miRNAs 及其靶基因在不育系和保持系之间的差异表达水平调控甘蓝花粉发育和雄性不育。项目的执行为明确miRNA对甘蓝雄性不育的基因表达调控作用提供了依据，并为调控作物花粉育性，推动杂种优势的广泛利用奠定基础。