水稻FYVE结构蛋白基因MS2调控花粉育性的分子机制研究

Male sterility is a fundamental question in biological research, as well as a key factor determining yield and utilization of heterosis in crop production. So far, numerous male sterility genes have been cloned in rice, yet the regulatory mechanism and network remain unclear. By screening from an irradiation-induced mutant library derived from variety 9311, we identified a male sterile line ms2 with abnormal vacuole development in pollens, while the female fertility and agronomic traits are normal, which exhibits promising potentials of the gene in breeding practice. Our preliminary research has mapped MS2 to a novel FYVE gene with potential phospholipids affinity in chromosome 2. We hypothesized that MS2 regulates the phospholipids metabolism on vacuole membrane, and eventually affect pollen fertility. This proposal aims to clone and functionally characterize male sterile gene MS2. Functional complementary test will be conducted to verify its biological function in rice. The expression profile and subcellular localization of MS2 will be investigated. Resin section and electronic microscopy techniques will be employed to cytologically characterize the ms2 pollen abortion and vacuole development. phospholipids affinity assay and protein-protein interaction assay will be conducted to find out the lipids and proteins interactive with MS2. Moreover, we will test the phospholipid metabolites contents in ms2 anther, and attempt to figure out the substrates and potential biochemical reactions catalyzed by MS2. Finally, we expect the outcome of this project will help us to elucidate the mechanism underlying the MS2-regulated male sterility. This study will greatly enhance our understanding of rice reproductive development, and provide useful gene resource for rice hybrid breeding.

雄性不育是生命科学研究的基础问题，也是决定作物产量和杂种优势利用的基础。水稻中已有大量的雄性不育基因被克隆，但调控机理和网络还不明晰。我们筛选得到了一个败育单株ms2，其花粉液泡发育异常，育性完全丧失，但雌配子育性和重要农艺性状无明显变化。图位克隆结果显示MS2位于第二染色体，编码一个具有磷脂亲和性的FYVE结构蛋白基因，且遗传互补能成功恢复育性。我们推测MS2调控液泡膜的磷脂代谢，最终影响花粉育性。本研究旨在克隆MS2基因，验证其生物学功能；检测MS2的时空表达模式和蛋白的亚细胞定位；确认ms2的雄性不育基础；鉴定MS2的互作蛋白。同时，我们还将检测ms2花药中的磷脂类代谢成分的变化，探索MS2的亲和磷脂类型、作用底物以及调控的代谢途径；最终期望能明晰MS2调控水稻雄配子发育的分子机制。本研究的实施将丰富我们对水稻育性发育的调控机制的了解，并为创制新的水稻雄性不育系提供优良的基因资源。

水稻是最重要的粮食作物之一，育性是决定水稻产量的关键因素，雄性不育资源也是水稻杂种优势利用的重要材料。我们前期筛选得到了一个败育单株rms2，其花粉液泡发育异常，育性完全丧失，但雌配子育性和重要农艺性状无明显变化。图位克隆结果显示RMS2位于第二染色体，编码一个具有磷脂亲和性的FYVE结构蛋白基因，且遗传互补能成功恢复育性。我们推测RMS2调控液泡膜的磷脂代谢，最终影响花粉育性。在此基础上，本研究针对rms2开展了表型观察、基因克隆、转录调控等工作，获得如下研究结果：.等位基因突变体表型与rms2一致，而遗传互补可将rms2育性回复正常，表明LOC_Os02g18870为RMS2基因。.细胞学观察显示rms2花药壁组织的绒毡层和中间层不降解，角质层和乌氏体形成滞后，花粉外壁的外壁内层不连续，小孢子的中央液泡发育缺陷。.RMS2主要在早期花药发育过程中转录，在绒毡层和小孢子中表达，而在其他组织中的转录水平较低。RMS2蛋白定位于细胞内质网中。.RMS2对通用底物p-nitrophenyl butyrate具有水解酶活性，rms2中酶活性显著降低。代谢组分析显示，RMS2突变导致16种脂质成分和其他代谢物的含量发生了显著变化，表明RMS2可以通过调控花药中的脂质代谢控制花粉育性。.水稻雄性育性关键调控因子UDT1和 PTC1可以与RMS2启动子上含E-box顺式结构的区域结合，并能在原生质体中激活RMS2的转录。另外RMS2在udt1、ptc1突变体幼穗中的转录水平相比野生型明显下降，表明RMS2可能是水稻雄性育性调控网络的关键节点。.本研究克隆了一个调控雄性育性的脂肪酶基因RMS2，其通过调控花药中的脂质代谢，影响花药壁、花粉壁的发育过程和液泡形态变化，从而决定花粉育性。为FYVE酶家族在植物生殖发育中的功能提供了新的思路，并为杂交水稻育种提供了潜在的不育基因资源。