水稻RING泛素连接酶介导的抗逆机制

The ubiquitin degradation pathway has been involved in plant defense signaling, and studies have shown that RING ubiquitin ligases regulate biotic and abiotic stresses in rice. The function of a U-box ubiquitin ligase OsPUB57 has been analyzied in our group, and another RING-type ubiquitin ligases (RING6) was picked up for functional studies in this project. We have obtained knockout and enhanced mutants for RING6, expressed and purificated RING6 in vitro, determined its ubiquitin ligase activity, and generated overexpression or RNA silencing transgenic lines for RING6. In this project, we will analyze these transgenic and mutant lines for their responses to biotic ( rice blast /bacterial blight ) or abiotic stresses ( drought/salinity). In addition, we will isolate and clone RING6-interacting protein genes using the yeast two-hybrid system, identified potential ubiquitinated substrates of RING6, determine subcellular localization of RING6 and its interacting proteins. These data will be useful to explore RING6-mediated resistance mechanisms against biotic and abiotic stresses.

泛素蛋白降解途径参与植物防御信号传导，已有研究表明RING泛素连接酶参与水稻生物胁迫和非生物胁迫调控。我们前期研究了一个U-box泛素连接酶OsPUB57的功能,本项目将对另一个RING型泛素连接酶（RING6）展开重点研究。我们已获得了RING6的基因敲除和增强突变体，体外表达及纯化了RING6蛋白以及确定了其泛素连接酶活性，并获得了RING6的超表达和RNA沉默转基因株系。本项目将分析这些转基因株系及突变体材料的抗生物胁迫（稻瘟病及白叶枯病）及非生物胁迫（干旱及盐碱）状况，利用酵母双杂交体系，分离和克隆水稻泛素连接酶RING6的互作蛋白基因，筛选可能的泛素化底物，并研究RING6及其互作蛋白的亚细胞定位，探索RING6介导的抗逆机制。

水稻是世界上最重要的粮食作物之一。由稻瘟菌（Magnaporthe oryzae）引起的稻瘟病（Rice blast）是威胁水稻产量最严重的真菌性病害，严重爆发时可引起水稻大幅减产。如何有效抑制稻瘟菌的致病性并增强水稻自身的抗病性是当前稻瘟病与水稻互作研究的主要焦点。本研究从粳稻品种日本晴中克隆了一个受稻瘟病诱导的新的编码RING-finger结构域的泛素连接酶基因OsBIRP1（Blast-Induced RING-finger Protein 1），即申请书中的OsRING6基因。通过表达分析及表型鉴定，我们认为OsBIRP1主要参与水稻稻瘟病的调控，并获得了以下主要研究结果：.1. OsBIRP1在水稻细胞的细胞核及细胞质中都有表达。.2. OsBIRP1可以被亲和性稻瘟菌和chitin诱导表达。.3. 接种稻瘟菌的表型分析发现，OsBIRP1-ACT激活突变体和OsBIRP1过表达材料都更加感病；而osbirp1 CRISPR材料则更加抗病，这表明OsBIRP1作为一个负调节因子参与了水稻对稻瘟病的调控。.4. 荧光素酶互补实验及免疫共沉淀分析发现，OsBIRP1在体内能与其自身相互作用而形成同源二聚体。.5. OsBIRP1具有体外自身泛素化活性，其点突变蛋白OsBIRP1 H273R能消除自身泛素化活性，说明OsBIRP1的体外自身泛素化活性依赖于第273位的组氨酸。.6. 26S蛋白酶体抑制剂MG132处理能增加OsBIRP1的积累，且RING-finger结构域的点突变蛋白OsBIRP1 H273R和OsBIRP1 C271W也变得更加稳定，这表明OsBIRP1蛋白的稳定性受到26S蛋白酶体途径的调控。.7. 通过蛋白组学分析，筛选得到了3个同时在OsBIRP1-ACT激活突变体和osbirp1-ko突变体材料中呈现差异表达的蛋白。