水稻F-box与PcG蛋白互作调控胚乳细胞周期的分子机理

Cell divison and diffrenciation in endosperm determine seed development in cereal crops. However, the signal factors and molecular mechanisms regulating endosperm cell cycling remain unclear. Our previous analysis suggested that down-regulation of rice polycomb group (PcG) gene OsiEZ1 lead to increasing number of endosperm cells. In addition, OsiEZ1 interacted with OsRBR1, a suppressor in RBR-E2F mediated cell cycling pathway, indicating that OsiEZ1 might be involved in exiting of cell cycling. Another interacting protein of OsiEZ1, OsFBO1, interacted with SCF E3 complex. It was expressed specifically in endosperm, and the knock-down mutant of OsFBO1 showed decreasing number in endosperm cells. Based on these results, we infer that OsFBO1 might maintain endosperm cell cycling by degradation of OsiEZ1.. In this work, we intend to construct the double mutant of OsFBO1 and OsiEZ1, OsiEZ1 and OsE2F1. And the cell cycling phenotypes in single and double mutant plants will be investigated, in order to determine their genetic relationship. We plan to make clear if OsiEZ1, OsRBR1 and OsE2F1 form three protein complexes, which would provide clues in the potential relationship of OsiEZ1 and RBR-E2F. In order to understand the molecular mechanisms regulating cell cycling, how the differential expression of OsiEZ1 affected the expression of cell cycling-related genes and variation of H3K27me3 levels of the targent of OsE2F1 will be analyzed. We will also analyzed the protein level of OsiEZ1 in fbo1 mutant, and under protease inhibitors, which will provide confime data in understanding the mataining mechinsm of cell cycling by OsFBO1 via OsiEZ1. Furthermore, the dynamic distributions of OsFBO1 and OsiEZ1 will be investigated, which can reveal the regulation mechanisms of maintaining and exiting of cell cycling. Our research will uncover the molecular mechanism of cell cycling regulation in rice endosperm cells, which has significant implication in rice genetic improvement.

胚乳细胞分裂时间影响谷类作物种子大小，但其细胞周期上游调控机制尚不明确。前期研究发现水稻PcG基因OsiEZ1表达下调致胚乳细胞数增多，且iEZ1与细胞周期RBR-E2F的抑制子OsRBR1互作，可能与细胞周期退出有关；另一互作蛋白OsFBO1还与SCF E3泛素复合物成员互作，其突变体胚乳细胞数减少，可能降解iEZ1以维持细胞周期。本项目拟构建iEZ1分别与FBO1和E2F1的双突材料，观察单突及双突植株的细胞周期表型，确定FBO1、iEZ1、RBR-E2F的上下游关系。研究iEZ1对细胞周期基因表达及E2F1靶基因H3K27me3修饰的影响，解析iEZ1调控RBR-E2F途径的分子机制。检测fbo1及蛋白酶抑制剂处理后iEZ1蛋白水平变化，分析FBO1调控iEZ1的机制。并研究FBO1和iEZ1的时空分布与细胞周期维持和退出的关系。以揭示这两个互作蛋白调控水稻胚乳细胞周期的分子机理。

谷类作物胚乳细胞数目和大小决定营养物质的积累，是种子产量形成的基础。为保障胚乳细胞数目和大小的协调发展，必然存在复杂精巧的调控机制，以调控胚乳细胞周期的维持和退出。本项目主要解析水稻PcG和SCF类2个互作蛋白调控胚乳细胞周期的分子机制。研究结果显示，PcG基因OsiEZ1下调表达和超量表达均导致水稻籽粒减小、千粒重降低。其下调表达增加胚乳细胞数目、减小胚乳细胞大小；超量表达致胚乳细胞化时间提前使细胞数量减少。超表达家系胚乳细胞核12C值出现的时间比野生型早， RNAi中12C值的胚乳细胞量少、且出现时间比野生型晚。OsiEZ1在胚乳细胞由分裂向分化转变的时期高表达，在淀粉胚乳中央区域的表达先减弱，在糊粉层区域的表达维持到发育更晚时期。互作实验显示OsiEZ1与RBR-E2F在体内和体外形成OsiEZ1-RBR-E2F复合物，且RBR-E2F途径的OsE2F1-3、OsORC2、OsMCM1-7和细胞复制相关基因OsCDKA1、OsCDKA2和OsCCS52A的表达受OsiEZ1负调节。其表达下调导致OsORC2和OsMCM7及基因组水平H3K27me3修饰降低。OsiEZ1-RNAi × OsE2F2-RNAi杂交后代的表型类似于OsE2F1-RNAi。因此，我们认为OsiEZ1通过RBR-E2F途径调控水稻胚乳细胞退出分裂周期进入核内复制。OsFBO1编码水稻F-box蛋白，在花后5-21天的胚乳中表达量最高。OsFBO1纯合突变体和RNAi转基因家系的胚乳细胞数量较野生型显著减少，超表达家系胚乳细胞数量与野生型差异不显著。OsFBO1表达下调导致细胞周期和储藏物质相关基因的表达下调。酵母双杂交、BiFC和Pull-down实验结果显示OsFBO1与OsiEZ1在体内和体外均能互作，非细胞体外降解实验表明，OsiEZ1蛋白的降解依赖于蛋白酶体系统。生化实验结果显示OsFBO1与水稻中的Skp1同源蛋白互作。将OsE2F2的cDNA全长分别在OsFBO1的启动子和ubiquitin启动子的驱动下转入osfbo1中，转基因植株胚乳细胞周期表型有部分回复。已有结果表明OsFBO1参与形成SCF E3泛素连接酶复合体，通过降解其靶蛋白OsiEZ1参与水稻胚乳细胞周期调控。本研究不仅解析了水稻胚乳细胞周期维持和退出的分子机制，也为水稻产量的分子育种提供了新的基因资源。