OsCUL3a介导的水稻程序性细胞死亡与广谱抗病性机制研究

OsCUL3a is an E3 ubiquitin ligase which was cloned from a lesion mutant lm-ZH in ZH11 background and negative regulation of lesion mimic phenotype. This lesion mimic phenotype is related to leaf position and development stage, and can enhance the broad-spectrum resistance to various rice blast species at the adult stage of lm-ZH (oscul3a) mutant. Using wild type variety ZH11 and the lm-ZH (oscul3a) mutant, the proteomic analysis was carried out using the top second leaf before and after the formation of lesion mimic, 4623 and 4572 functional proteins were quantitatively identified, respectively. The functional annotation of proteins showed that the differential proteins were mainly located in chloroplast and cytoplasm, and involved in the biological processes of metabolism and catalysis. The phenyloropanoid pathways involved in the synthesis of important anti-adverse secondary metabolites such as SA (salicylic acid) and anthocyanin were significantly enhanced after the occurrence of lesion mimic. Based on the previous research work, the subsequent research will be focused on following aspects by gene knockout and genetic analysis method: (1) the mechanism of OsCUL3a negatively regulates the Phenyloropanoid pathway and causing the mutant’s lesion mimic formation and improving the broad-spectrum resistance to disease. (2) Mutations in OsCUL3a inhibit and induce high expression of many functional proteins and make unfavorable changes in some important agronomic traits. So how OsCSN5, OsRUB1/2, OsCAND1 and OsRBX1a fine-regulate the ubiquitin modification biological function of OsCUL3a and its relationship with lesion mimic formation need further elucidation.

OsCUL3a负向调控类病斑表型，其特征与叶位和发育时期有关，类病斑表型能增强lm-ZH(oscul3a)对稻瘟菌的广谱抗病性。利用野生型ZH11和类病斑突变体lm-ZH(oscul3a)，在类病斑形成前和形成后取倒数第二叶片进行蛋白组分析，分别鉴定出功能蛋白4623和4572个；差异蛋白主要定位在叶绿体和胞质内，参与代谢和催化等生物学过程，其中Phenyloropanoid代谢途径在类病斑出现后显著增强。基于此，后续研究将通过遗传分析的方法去验证：（1）OsCUL3a参与调控Phenyloropanoid代谢途径，影响次生代谢物细胞内和组织间转运，与类病斑表型和广谱抗病性的关系；（2）OsCUL3a的突变影响许多功能蛋白的表达，导致很多重要农艺性状变化，所以OsCSN5，OsRUB1/2，OsCAND1和OsRBX1a如何精细调控OsCUL3a的生物学功能，及其对类病斑表型的影响。

病害是水稻安全生产的重要制约因素，深入挖掘抗病相关基因，系统阐述水稻抗病的分子机制是开展水稻抗病分子育种，减轻水稻病害损失的根本举措。以免疫自激活造成的类病斑突变体为载体，为反向溯源性分离上游抗病基因或正向深入研究调控水稻免疫的分子机制提供了极佳的突破口。我们的前期研究结果显示，OsCUL3a功能缺失突变体表现依赖于OsNPR1的细胞死亡和抗病性增强表型。通过本项目的实施，我们对OsCUL3a参与调控的次生代谢过程以及oscul3a突变体类病斑形成的生理生化与细胞学基础开展研究，同时通过抑制子筛选在遗传上初步探讨了oscul3a突变体细胞死亡表型的依赖因子及其调控机理，结果显示，oscul3a突变体类病斑的形成由叶片局部组织区域的H2O2积累和细胞死亡产生，然后逐渐扩展到整片叶片；oscul3a突变体叶片中H2O2积累动态的生理变化，使其在生长后期对稻瘟病和白叶枯病的抗性显著提高。透射电镜观察发现，oscul3a突变体叶片细胞中有大量脂质体积累，叶绿体中有大量嗜锇颗粒积累，脂质体和嗜锇颗粒的积累与脂类代谢有关；利用蛋白组学分析发现，差异表达蛋白主要定位在叶绿体和胞质中，参与细胞内代谢过程，使oscul3a突变体的脂类次生代谢途径增强，而碳氮代谢途径减弱，水杨酸含量在oscul3a突变体叶片中显著升高；同时发现另一增强的代谢产物香豆素具有稻瘟病菌生长抑制活性，可诱导水稻愈伤ROS产生；通过回复突变筛选到一个oscul3a的抑制子SC1，类病斑表型在oscul3a/sc1双突变体中得以完全恢复。SC1编码一个典型的CC-NBS-LRR类抗性蛋白，是水稻系统抗性的一个核心调控因子。以上结果可为OsCUL3a在水稻抗病性分子改良中的应用提供理论指导。