microRNA调控红麻细胞质雄性不育发生的分子机制研究

The cytoplasmic male sterile (CMS) lines played a crucial role in utilization the heterosis of crops. To date, little is known about the mechanism on the molecular basis of CMS. In the present study, the anthers of CMS and its maintainer lines were used for CMS mechanism investigating. Firstly, total RNA will be isolated to construct libraries for miRNA sequencing, and based on this to construct miRNA differential expression profiles. Secondly, we will use Degradome sequencing method to reveal miRNA target genes. The differentially expressed genes will be analyzed by GO annotation and KEGG pathway analysis. Thirdly, some of the pre-miRNA will be cloned after RT-PCR and Northern blot verification. These miRNAs will be silenced or over-expressed in kenaf to evaluate their function by Agrobacterium mediated transformation. The result, combined our present research, should reveal the mechanism of kenaf CMS and give some clues in elucidating the CMS of other crops.

细胞质雄性不育(CMS)是杂种优势利用的基础，本课题组率先发现并选育了红麻CMS系，但关于红麻CMS发生的机制研究尚鲜见报道。本研究以红麻CMS系和保持系为材料开展以下研究：首先分别分离其花药总RNA，进行miRNA测序分析，构建miRNA差异表达谱。然后利用降解组测序技术筛选miRNA靶基因，对靶基因进行GO功能注释和富集分析，并进行KEGG代谢通路分析，研究靶基因参与哪些生物学功能。克隆2~3个候选miRNA前体，构建干扰载体及过表达载体，导入可育材料中干扰或者过表达miRNA，获得转基因植株，鉴定与红麻CMS相关的关键miRNA。在转基因植株中，利用RT-PCR及Northern blot等手段验证miRNA及靶基因表达，分析转基因植株育性。结合上述研究结果，初步阐明miRNA调控红麻CMS发生的作用机制。

细胞质雄性不育(CMS)是杂种优势利用的基础，本课题组率先发现并选育了红麻CMS系，但关于红麻CMS发生的机制研究尚鲜见报道。本研究以红麻CMS系和保持系为材料，分别分离其花药总RNA，进行miRNA测序分析，构建miRNA差异表达谱；发掘283个已知microRNA和46个新microRNA，共筛选到67个差异表达microRNA，qRT-PCR验证结果与测序基本一致；通过降解组测序发现67个差异表达microRNA参与调控189个靶基因的调控。通过生物信息学分析，靶基因参与cellular component biogenesis, growth, reproductive process, developmental process, biological regulation, metabolic process, organelle, transporter activity, and catalytic activity被高度富集；为了明确microRNA对靶基因的调控作用，我们对红麻不育系和保持系花药，同时对miRNA与其靶基因表达关系进行了RT-PCR表达分析，结果表明：大部分miRNA与靶基因的表达量呈现负相关，部分miRNA与其靶基因表达量也会表现出正相关关系，表明除了miRNA外，还存在其他的调控因子。另外，克隆了多个microRNA基因，构建Crispr/Cas9载体和过表达载体，并利用农杆菌介导转化了烟草对其功能进行了验证。通过分析差异表达microRNA、靶基因调控，关键microRNA的功能解析，我们提出了miRNA调控红麻细胞质雄性不育发生的代谢通路模型。其中激素信号通路、信号转导调控、基因表达调控、糖酵解与能量代谢通路、物质转运、蛋白质修饰与代谢调控以及花发育调控相关的代谢过程在红麻细胞质雄性不育发生中发挥重要的作用。