水稻OsSAP5基因调控种子灌浆成熟期高温耐性的分子机制

灌浆期热害是影响水稻种子生产的主要气象灾害之一。本项目前期研究发现，亚精胺Spd可调控OsSAP5参与水稻种子灌浆期的高温耐性，ossap5突变体水稻种子的耐热能力显著下降。但OsSAP5是否具有E3泛素连接酶活性，并通过介导其互作蛋白的泛素化降解，诱导热激转录因子的表达，提高水稻种子成熟期的耐热性，还是存在其他调控机制尚不清楚。因此，本项目拟利用水稻ossap5突变体及过表达OsSAP5转基因株系，分析灌浆期高温逆境下，该基因对种子发育的调控及种子质量的影响；鉴定OsSAP5的E3泛素连接酶活性，采用CoIP-MS技术寻找OsSAP5的互作蛋白，利用生化与分子技术验证蛋白互作，并构建真实互作蛋白的突变体，分析其作用功能；进一步结合转录组分析，探讨OsSAP5参与水稻种子高温胁迫应答的调控网络，研究结果将为阐明水稻种子高温响应机理及高活力水稻种子生产和选育耐热水稻品种提供理论与技术支撑。

High temperature stress is one of the main meteorological disasters affecting rice seed-production. In our previous study, OsSAP5 regulated by spermidine was found to be involved in the high temperature tolerance of rice seed during grain-filling stage. The significant lower heat-resistance was observed in the rice mutant of OsSAP5. However, it is unclear whether OsSAP5 has the activity of E3 ubiquitin ligase, and induces the expressions of heat shock transcription factors by mediating ubiquitination degradation of the proteins it interacts with to improve the heat resistance of rice seeds, or whether there are other regulatory mechanisms. In this study, the rice ossap5 mutants generated by CRISPR/Cas9 approach and over-expression transgenic lines of OsSAP5 will be used to further clarify the regulation of OsSAP5 on seed development and seed quality under high temperature stress during the grain-filling stage. The E3 ubiquitin ligase activity of OsSAP5 will be identified. Meanwhile, the interaction proteins of OsSAP5 will be screened through the CoIP-MS technology, and these candidate proteins will be verified through the biochemical and molecular approaches. The functions of the above confirmed proteins will be further validated using their constructed mutants in rice. Finally, the potential molecular mechanism of OsSAP5 involving in the regulation of rice response to high temperature stress will be discussed with the transcriptome analysis. The results will provide theoretical basis and technical support for elucidating the response mechanism of rice seed to high temperature stress during seed maturation, producing rice seeds with high vigor and breeding heat-resistant rice varieties.