一个CCCH锌指蛋白调控水稻雄性生殖发育中胼胝质代谢的研究

Male reproductive development is an essential biological process for flowering plants and is important for crop seed production. During the past decades, a number of the related genes involved in male reproductive development have been isolated, but there are few researches on CCCH zinc finger protein and callose metabolism. In previous study, we isolated a new male sterile mutant ms18 from our rice mutant library. The ms18 mutant shows abnormal callose metabolism in anther development. The MS18 gene was identified through map-based cloning, which is encoding a CCCH zinc finger protein. In this project, we will first confirm the candidate gene through genetic transformation analysis, then compare the morphological differences between the mutant and the wild type and detect the expression pattern of the MS18 gene. Subsequently, through the screening of downstream factors, we will explain the molecular mechanism of MS18 regulating the callose metabolism, and clarify the role of the CCCH zinc finger protein in rice male reproductive development. This study will not only help people furtherly understand the regulation network of rice male reproductive development, but also provide the prerequisite for using of rice heterosis. Overall the research has important theoretical and practical value.

雄性生殖发育是开花植物的重要生物学过程，同时也是保障农作物种子产量的重要农艺性状。目前关于水稻雄性生殖发育调控基因的研究报道很多，但却鲜有关于锌指蛋白及胼胝质代谢调控网络的研究。我们前期的研究从γ-射线诱变的水稻突变体库中筛选了一个花粉败育的雄性不育突变ms18，通过图位克隆得到了该发育相关基因MS18，该基因编码一个CCCH锌指蛋白，ms18突变体的胼胝质的代谢出现异常。本项目拟通过遗传转化分析进一步确定候选基因，比较突变体和野生型在形态学上的差异及检测MS18基因的表达时空性，探讨MS18基因调控花药发育的时期和位置；通过筛选下游因子解释MS18蛋白调控胼胝质代谢的分子机理，阐明CCCH锌指蛋白在水稻雄性生殖发育中的作用机理。本项目研究不但有助于人们进一步的理解水稻雄性生殖发育的调控网络，也为水稻杂种优势的利用提供前提条件，因此本项目的研究具有重要的理论价值和实践意义。

雄性生殖发育是水稻的重要生物学过程，也是杂交水稻应用的基础。在国家基金青年基金项目（31600242）的资助下，本项目三年来深入研究了MS18对水稻雄性生殖发育的调控的分子机理，完成了预期的研究内容，并取得了如下研究成果：1）利用CRISPR/Cas9敲除Os06g0638000，确定MS18基因；2）通过突变体和野生型花药形态学的分析发现，MS18主要影响了花药壁层的降解和胼胝质的积累；3）亚细胞定位和酵母激活实验证实MS18编码的CCCH蛋白具有转录激活的活性；4）利用转录组测序分析和体外启动子结合分析证实MS18调控了OsGA20ox3基因和OsUGP2基因的表达，进而影响了花药壁层的降解和胼胝质的积累。本研究对其他类型的雄性生殖发育调控基因具有重要的借鉴作用，同时为利用CRISPR/Cas9基因组编辑创制雄性不育系提供了新的靶基因。本项目执行期共申请专利1项，在投稿论文1篇。培养副研究员1人，博士后2名，本科生2人圆满完成了既定目标。