小白菊内酯对水稻白叶枯病菌的氧化胁迫机制研究

Lack of control agents against rice bacterial blight and significant bactericide resistance of Xanthomonas oryzae pv. oryzae (Xoo) are urgent problems in China at present. In our previous studies, botanical active component parthenolide (PTL) was found very effective against Xoo, indicating its potential as a bactericide; PTL induced ROS accumulation in Xoo, and scavenge of ROS led to significant decrease of bacterial inhibitory activities, suggesting PTL’s mechanism of action is associated with oxidative stress. In this study, Xoo will be used as target organism, in which role of ROS in PTL’s bacterial inhibitory activities will be investigated. Also, the anti-oxidant system inhibitory activities of PTL will be analyzed. After that, biotin-streptavidin system (biotinylated PTL was already synthesized) will be employed to capture oxidative stress-related interaction proteins of PTL, and interaction validation will be conducted after prokaryotic expression and purification of candidate proteins. Moreover, gene knockout and over-expression of validated crucial proteins will be subsequently carried out and the sensitivity of mutants to PTL will be analyzed to figure out target-related proteins. This study can partially explain PTL’s mechanism of action against Xoo, which may scientifically guide further utilization and structure optimization of PTL.

目前，我国水稻白叶枯病防治药剂匮乏、抗药性问题显著。申请人前期研究发现植物源活性成分小白菊内酯对水稻白叶枯病菌具有强烈的抑制作用，是一种潜在的防治药剂；该化合物能够诱导水稻白叶枯病菌积累活性氧，且清除活性氧后其抑菌活性大幅下降，推测其作用机制与氧化胁迫有关。因此，本项目拟以水稻白叶枯病菌为靶标生物，分析活性氧在小白菊内酯抑菌活性中的作用，并测定小白菊内酯对病原菌抗氧化系统的抑制活性；合成生物素标记的小白菊内酯，引入生物素-链霉亲和素系统来捕捉与小白菊内酯互作的氧化胁迫相关蛋白，并在原核表达纯化后进行互作验证；对其中重要的互作蛋白进行基因敲除、过表达及突变体药敏性分析，找出靶标相关蛋白。本研究能初步解析小白菊内酯对水稻白叶枯病菌的作用机制，从而为小白菊内酯的科学开发利用及后续结构优化提供理论依据。

水稻白叶枯病是由水稻黄单胞菌水稻致病变种引起的重要细菌病害。目前，我国水稻白叶枯病防治药剂匮乏、抗药性问题显著。申请人前期研究发现植物源活性成分小白菊内酯对水稻白叶枯病菌具有强烈的抑制作用，是一种潜在的防治药剂；该化合物能够诱导水稻白叶枯病菌积累活性氧，且清除活性氧后其抑菌活性大幅下降，推测其作用机制与氧化胁迫有关。因此，本项目以水稻白叶枯病菌为靶标生物，从生理生化角度探究了小白菊内酯的作用方式，发现小白菊内酯通过大幅降低菌体GSH水平，扰乱GSH/GSSG平衡，造成氧化胁迫从而发挥抑菌活性，并且氧化胁迫在其抑菌活性中至关重要；对小白菊内酯抑菌活性基团进行了结构修饰，并进行构效关系研究，发现环外双键是其抑菌的关键活性基团，而在环外引入氨基能够增加其抑菌活性；合成了生物素标记的小白菊内酯，通过生物素-链霉亲和素系统捕捉到一些潜在互作蛋白，对其进行原核表达纯化及互作验证，发现水稻白叶枯病菌呼吸链上的NuoF亚基和SdhB亚基是小白菊内酯的互作蛋白；对这两个重要的互作蛋白进行同源模建、分子对接、基因敲除、过表达及突变体药敏性分析，发现它们是小白菊内酯的重要靶蛋白。本研究取得的成果初步解析了小白菊内酯对水稻白叶枯病菌的作用机制，从而为小白菊内酯的科学开发利用及结构优化提供了理论依据。