新型大环内酰胺化合物对香蕉枯萎病原菌作用机理研究

Due to the difficulty of research and development on the chemical pesticide, it is an effective and efficient way to find biological pesticide from the nature, especially microbial pesticide. Early, actinomycete, Streptomyces aureoverticillatus, was isolated from soil in Hainan, which showed significantly controlling effect against fusarium oxysporum f. sp. Cubenserace 4. Also, the strain exhibited excellent broad spectrum activities against other more than 20 kinds of target fungi, at the same time, the strain promoted the banana plant growth. And then, isolation and purification of the fermentation metabolites of the strain, a novel 22 atom lactam compound (YY3) was yield. The compound YY3 had obvious antifungal activity, but it is unclear about the model of action of this compound. Thus, in this project, subcellular localization of YY3 against fusarium oxysporum will be analyzed by colloidal immunization methods; The ultramicro morphology and the cellular structure of fusarium oxysporum will be observed under electronic microscope; The substance metabolism and energy metabolism of YY3 against fusarium oxysporum will be determined by physiological and biochemical methods; The transcriptome of fusarium oxysporum will be studied using molecular techniques; At the same time, in order to find out the effect on the plant defense system of the compound YY3, the research of inducible resistance of the compound YY3 on the banana plant will be carried out. After a series of exploration, we want to disclose the model of action of the novel large ring lactam YY3, and lay a foundation for the application of the compound.

随着化学农药研发的难度越来越大，从自然界中寻找生物农药，特别是微生物农药将是一种有效而快捷的途径。前期从海南土壤中分离获得一株金黄垂直链霉菌（Streptomyces aureoverticillatus）。该菌对香蕉枯萎病有显著防效，对其它20余种靶标真菌抑菌谱广，同时具有促生作用。随后分离纯化发酵代谢产物，得到一种22原内酰胺化合物（YY3），YY3有显著的抑菌活性，但其作用机理目前还不清楚。为此，本项目采用免疫胶体金法研究YY3在香蕉枯萎病菌中亚细胞定位；利用电镜技术观察YY3对香蕉枯萎病菌的超微形态及细胞结构的影响；采用生理生化方法测定YY3对香蕉枯萎病菌的能量代谢、物质代谢影响；借助分子手段分析YY3对香蕉枯萎病菌转录组影响；同时为了探明该化合物对植株防御系统的影响，开展YY3对香蕉植株诱导抗性研究。经过系列探索，以期揭示该新型大环内酰胺化合物的作用机理，为该化合物的应用奠定基础

香蕉枯萎病是由尖孢镰刀菌古巴专化型（Fusarium oxysporum f. sp. Cubeme）侵染引起，是香蕉产业毁灭性病害。化学防治病害伴随着污染大、易残留、易产生抗药性等问题，使得人们对生态平衡和绿色健康概念越来越重视，生物防治措施在农业生产中得到了前所未有的关注，从微生物中寻找具有杀菌活性的物质具有重要的意义。项目前期研究发现链霉菌HN6对香蕉具有很好的防病促生作用，其主要活性物质为大环内酰胺类化合物YY3。为明确该化合物的作用机理，我们以香蕉枯萎病原菌为靶标菌，采用免疫金亚细胞定位技术、生理生化方法、分子手段以及荧光分析方法从细胞水平、生理生化水平以及分子水平研究YY3对靶标菌的影响。结果发现，YY3主要作用的细胞器为线粒体；经YY3处理后靶标菌细胞壁变薄，线粒体肿胀；其对靶标菌的生物合成和生物氧化均存在明显的影响，尤其对线粒体复合酶III存在明显的抑制作用；在20.8 μg/mL浓度下，YY3孵育靶标菌菌丝4 h，细胞质内钙离子浓度显著增加；孵育6 h，靶标菌内出现活性氧迸发，线粒体膜电位显著降低；孵育12 h，细胞凋亡检测为阳性；同时YY3对靶标菌转录水平的影响提示，磷脂酰肌醇信号通路显著被影响。综合以上结果我们推测YY3作用机理为，YY3通过作用靶标菌内的磷脂酰肌醇信号系统使得细胞内钙离子显著增高，线粒体对Ca2+的大量摄取导致线粒体基质肿胀，同时造成ROS的大量生成，使得线粒体膜电位降低、线粒体膜破损，线粒体内细胞凋亡因子释放，线粒体介导的细胞凋亡通路被激活，诱导靶标菌细胞凋亡。