油菜内源抗菌肽BnPRP1对核盘菌的抑菌机理研究

Antimicrobial peptides act as the first line of defense against pathogens and pests, which have effective microbial inhibitory activities. Sclerotinia Stem rot disease is caused by Sclerotinia sclerotiorum (Lib.) de bary. This worldwide disease is the most severe disease of Brassica napus in China. The antimicrobial peptide, BnPRP1, screened from Brassica nupus, is the first and hitherto the only one proline-rich antimicrobial peptide in plant. In our previous work, we have identified that BnPRP1 has excellent inhibitory activity on S. sclerotiorum both in vivo and in vitro. However, the mechanisms of action on S. sclerotiorum are still unclear. In this project, we attempt to identify the targets of BnPRP1 in S. sclerotiorum by proteomic sequencing, immune affinity purification and LC-MS/MS analysis. And then, we will validate the key roles of the BnPRP1 targets in S. sclerotiorum metabolic pathways through genetic transformation. Finally, we will reveal the mechanisms of action against S. sclerotiorum. Our project will supply the theoretical basis for designing new targeted agents for controlling S. sclerotiorum, and lay a foundation for developing a new green strategy for controlling Sclerotinia Stem rot disease.

抗菌肽是宿主非特异性防御系统的一部分，具有广谱抗菌活性。油菜菌核病由核盘菌（Sclerotinia sclerotiorum（Lib.）de bary）引起，是一种世界性真菌病害，同时是我国油菜生产中最主要的病害。油菜内源抗菌肽BnPRP1是植物体内鉴定的首个也是目前唯一一个富含脯氨酸的抗菌肽。BnPRP1无论在体内还是体外均对核盘菌具有显著的抑菌效果，然而其作用机制尚不明确。本项目拟通过蛋白组学、免疫亲和纯化技术及质谱技术分离鉴定核盘菌中与BnPRP1互作的靶标，并通过核盘菌的遗传转化验证靶标对核盘菌生长代谢的影响，最终揭示BnPRP1对核盘菌的作用机制。该项目BnPRP1独特的抑菌机制的解析能够为设计全新的靶向药剂防治菌核病提供理论依据，为开发新型绿色安全环保的菌核病防治策略奠定基础。

抗菌肽是宿主非特异性防御系统的一部分，具有广谱抗菌活性。油菜菌核病由核盘菌（Sclerotinia sclerotiorum（Lib.）de bary）引起，是一种世界性真菌病害，同时是我国油菜生产中最主要的病害。油菜内源抗菌肽BnPRP1是植物体内鉴定的首个也是目前唯一一个富含脯氨酸的抗菌肽。BnPRP1无论在体内还是体外均对核盘菌具有显著的抑菌效果，然而其作用机制尚不明确。本项目通过流式细胞仪对BnPRP1处理的核盘菌子囊孢子细胞膜的完整性进行检测，结果显示随着BnRPP1浓度的升高，PI标记细胞百分比并未明显上升，因此我们推测BnPRP1不是通过破坏核盘菌的细胞膜而发挥抑菌活性。之后本研究通过pull-down技术筛选到BnPRP1在核盘菌中的候选靶标有两个，分别为actin和含有WD40结构域的假定蛋白。之后核盘菌遗传转化及油菜接种实验表明BnPRP1能够通过结合核盘菌体内的靶标，降低核盘菌活性，从而达到抑菌的作用。该项目BnPRP1独特的抑菌机制的解析为设计全新的靶向药剂防治菌核病提供理论依据，为开发新型绿色安全环保的菌核病防治策略奠定基础。