控制水稻花药发育PHD-finger蛋白的功能研究

The application of rice male sterility plays an important role in the process of rice yield improvement. Most male sterility is caused by the defective anther or pollen development. Therefore, the study about rice anther and pollen development is critical for practical application in rice breeding and understanding the molecular mechanism of male reproductive development. In rice, a PHD-finger protein, encoded by PTC1, is an important gene expression regulator, which plays an important role in anther development. However, the fundamental mechanism of PTC1 is unknown, and whether the PHD-finger protein regulates gene expression by interacting H3 histone protein is also unknown. Previously, we found a spontaneous male sterile mutant osms1, which is allelic to ptc1. In this project, we plan to use Pull Down and CoIP assays to analyze the interaction between OsMS1 and various methylated H3 histone, and then use west blot to analyze the interacted protein level between wild type and osms1, and use Chip-seq to genome-wide analyze the target genes of H3 histones which are interacted with OsMS1 and down-regulated in osms1, and analyze the relationship between down or up-regulated genes and histone methylation. The result will be helpful for understanding the fundamental mechanism of PHD-finger protein OsMS1, and for understanding the gene regulation network of anther development.

雄性不育的利用在水稻产量提高过程中做出了巨大贡献。大多数雄性不育突变体都是由花药或花粉粒发育异常所导致的，因此相关分子机理的研究对操控水稻育性以及了解雄性生殖发育的分子机理都具有非常重要的意义。水稻PTC1编码一个PHD-finger蛋白，作为一个至关重要的基因表达调控因子参与花药发育过程中。ptc1表现为绒毡层细胞降解延迟以及雄性不育。但作为表观遗传因子之一的PHD-finger蛋白是通过什么深层次的分子机制来调节基因表达，以及是否与组蛋白相互作用来调控基因表达，这些问题目前都不清楚。本项目拟以一个ptc1等位突变体（osms1）为研究材料，在探明与OsMS1具有相互作用的组蛋白甲基化类型后，用相应组蛋白特异抗体检测突变体中组蛋白甲基化修饰水平，确定OsMS1所影响的组蛋白修饰；再利用Chip-seq全基因组分析OsMS1影响的组蛋白甲基化情况，深层次阐明雄性生殖发育的分子机理。

本项目主要研究内容为PTC1调控花药发育的分子功能。主要结果如下： ptc1-1（SC-ms-2）突变体中PTC1启动子区域1036bp片段被52bp替换，导致PTC1在突变体中表达量极低。PTC1在小孢子时期的绒毡层和小孢子中特异表达，具有核内转录激活活性，并调控涉及信号通路等途径基因共797个。PTC1蛋白通过PHD finger结构域识别H3K4me3修饰。酵母双杂系统结果表明，PTC1与OsEMF2b相互作用。但在野生型和突变体花药材料并没检测到组蛋白甲基化修饰明显的变化。这可能是由于PTC1在绒毡层和小孢子中特异性表达所导致。因此任务书中拟开展的在花药中全基因组鉴定受PTC1影响位点的计划，没有进一步开展。转向从筛选恢复突变的角度对PTC1的功能进行研究。通过对PTC1在拟南芥同源基因突变体ms1-1回复突变体筛选，目前共获得5份育性明显恢复的恢复子。我们结果为PTC1功能进一步深入研究提供良好的信息和基础材料。