芽孢杆菌抗真菌肽P852抑制尖镰孢菌的作用位点及靶分子解析

Fusarium wilt disease is the main crop soil-born disease. Fusarium oxysporum （pathogen）could infect cucumbers, peppers, tomatoes, rice, wheat, cotton, alfafa, orchardgrass, tall fescue and other crops. This disease has caused heavy economic losses. However, current chemical controls are not effective. Biological control is an efficient and environment-friendly way to prevent fungal disease. Bacillus spp. can secrete antimicrobial peptides to inhibit the growth of pathogenic fungi. . In previous work, we had obtained an excellent biocontrol Bacillus strain L-H15. The antifungal peptides secteted by L-H15 plays an important role in biocontrol. Antifungal peptides have been purified to homogeneity by acid precippitaton, gel permeation chromatography, and reversed-phase high-performance liquid chromatography. Antifungal peptides’ structural features (molecular weight, amino acid composition, connection order) analyzed by FTIR, automatic amino acid analyzer, Matrix-assisted laser desoption ionization-time of flight mass spectrometry methods. We found a novel peptide P852 which could strongly inhibit the growth and reproduction of Fusarium oxysporum ，lead cell membrane and organelle damage and cell death. Its antifungal activity is superior to the fungal antibiotic amphotericin B. P852 has potential application value, but its antifungal mechanism remains to be revealed. . There are three main research contents in this project: (1) Study the physiological responses of Fusarium oxysporum to P852; (2) Determine antifungal peptide’s subcellular localization and action targets in Fusarium oxysporum cell by using P852-specific antibody combined with confocal laser scanning microscopy and transmission electron microscopy; (3) Identify and analyse P852 receptor molecule by using molecular hybridization, mass spectrometry and bioinformatics. The purpose is to clear the antifungal peptide’s action mode and mechanism from cellular and molecular levels. The results could provide scientific basis for elucidationg the action mechanisam of antifungal peptide P852 and for exploring new, safe, non-toxic and cost-effective biocontrol agents to reduce or take the place of chemical fungicides.

尖镰孢菌（Fusarium oxysporum）引起的镰刀枯萎病是粮食作物、蔬菜作物和饲草普遍发生的一种毁灭性真菌病害。我们研究发现解淀粉芽孢杆菌（Bacillus amyloquefaciens） L-H15分泌的新型抗真菌肽P852，能破坏病原菌细胞膜和细胞器，进而抑制其生长、繁殖，直至死亡，具有潜在的应用价值。本课题将在系统掌握尖镰孢菌对P852生理应答规律的基础上，利用P852特异性抗体结合免疫荧光和免疫电镜技术确定P852在尖镰孢菌细胞内的作用位点；通过分子杂交、质谱分析和生物信息技术等，确定并解析P852的受体分子。从细胞和分子水平深入解析抗真菌肽P852抑制尖镰孢菌的作用方式和途径，为研制新型、安全、高效抗真菌物质提供科学依据。

本课题以土传病害镰刀枯萎病的病原菌尖镰孢菌为指示菌，从细胞和分子水平揭示抗菌肽P852抑制尖镰孢菌的抑菌机制。结果显示，抗菌肽P852作用位点为细胞质膜和线粒体膜，该化合物可以诱导靶标菌产生氧化应激与细胞凋亡。结合前期研究结果推测其作用机理为：结合前期研究结果推测其作用机理为：抗菌肽P852进入靶标菌后，通过多靶点机制共同改变线粒体内外膜的通透性，在线粒体内逐渐富集，影响线粒体复合酶 III 的活性，阻断线粒体呼吸链电子传递，影响能量生成；同时对 GR 等抗氧化酶的抑制作用导致 ROS蓄积，促使线粒体通透性转换孔（MPTP）持续开放，导致Cyt c和其他分子从孔中释放，引发caspase-3依赖性途径诱导细胞凋亡。由于MPTP跨越线粒体的内膜和外膜，它的持续开放也会直接引起线粒体外膜通透（MOMP）和线粒体内膜通透（MIMP），进而破坏线粒体的结构，最终使得菌体死亡。该抑菌机制可为研制新型、安全、高效抗真菌物质提供科学依据。. 抗菌肽对植物镰刀枯萎病的防控试验表明，P852可通过增强植株抗氧化防御、抗真菌酶活性、以及诱导代谢产物异喹啉生物碱、甜菜碱和精氨酸的生物合成，恢复植物生长和发育，提升植物在尖镰孢菌侵袭下耐受性的机制。本研究揭示的抗菌肽抑病机理将为绿色农药的开发提供新的思路。.