植物源活性物质天名精内酯酮抑菌作用机理研究

Carabrone is one new agricultural fungicidal active compound obtained from Carpesium macrocephalum. Primary studies indicated that carabrone in vitro had strong activity against many phytopathogens, including Gaeumannomyces graminis, Colletotrichum lagenarium and Botrytis cinerea. With obvious uptake and translocation activities, carabrone exhibited well control effects against wheat take-all, powdery mildew, yellow rust, and gray mold in pot experiments. Abnormal changes of pathogen's cytoplasm and mitochondria were observed in electron microscopy tests. Carabrone exhibited no discernible effect on membrane permeability of G. graminis. In co-toxicity test, alicylhydroxamic acid (SHAM) enhanced carabrone's activity as well. Preliminary studies indicated that carbrone might mainly affect the biological oxidation processes of G. graminis. In this project, our research group plan to apply MARG (microautoradiography) with 14C in situ labeled carabrone and Immune colloidal gold technique with monoclonal antibody of carabrone to investigate the compound's subcellular localization in G. graminis, 2D gel electrophoresis coupled with autoradiograph to separate and identify target protein, physiological and biochemical methods to explore carabrone's effect on pathogen's mitochondrial dysfunction. The above research programs are necessary to be conducted to lay the foundation for approaching molecular mechanism of carabrone, a botanical fungicidal active compound, and provide a theoretical basis for biorational design and exploitation of new fungicides.

天名精内酯酮是从菊科天明精属植物大花金挖耳中分离得到的一种新农用抑菌活性化合物。系统测试表明，其对小麦全蚀病菌、黄瓜炭疽病菌和番茄灰霉病菌等多种病菌均具较强的离体抑制活性；对小麦全蚀病、白粉病和条锈病及番茄灰霉病等病害均具良好防效；前期形态学和生理生化研究表明，其可致病菌细胞质和线粒体等发生异常变化，对小麦全蚀病菌细胞膜通透性无明显影响，水杨肟酸对天名精内酯酮具明显的增效作用，说明其可能对病菌的生物氧化具明显影响。本项目拟合成天名精内酯酮14C标记物和单克隆抗体，结合采用显微放射自显影和免疫胶体金法进行天名精内酯酮亚细胞定位研究，并利用双向电泳结合放射自显影进行靶蛋白的分离、识别和鉴定；采用生理生化方法探讨其对病原菌线粒体功能的影响。旨在为进一步阐明植物源杀菌活性物质天名精内酯酮的抑菌作用分子机理和新型杀菌剂的生物合理设计及创制奠定基础。

天名精内酯酮是从菊科植物大花金挖耳中分离得到的一种新农用抑菌活性化合物，具有较大的产业化开发潜力，但其抑菌机理尚不明确。依据前期基础，本项目以小麦全蚀病菌为供试菌种，就其抑菌作用方式与机理进行了探讨，主要结果如下：.【1】天名精内酯酮具良好的内吸作用。以单子叶作物小麦和双子叶作物黄瓜为供试对象，药效试验和HPLC检测分析表明，天名精内酯酮可被单、双子叶作物的根部吸收并向上传导，但无明显的自上而下传导。该结果为天名精内酯酮的科学合理使用提供了理论支撑。.【2】荧光标记、免疫荧光和免疫胶体金技术3种方法的亚细胞定位表明，天名精内酯酮主要作用于病菌线粒体。.【3】天名精内酯酮可诱导靶标菌凋亡，原因为：该化合物可抑制病菌过氧化氢酶（CAT）和超氧化物歧化酶（SOD）活性，并致线粒体内谷胱甘肽过氧化物酶（GSH-PX）活性显著上升，使病菌不能及时清除活性氧，导致小麦全蚀病菌线粒体活性氧大量积累，从而激活线粒体介导的细胞凋亡途径，同时造成膜结构破坏、膜电位下降。.【4】天明精内酯酮对病菌线粒体生物氧化有显著影响。天明精内酯酮可抑制呼吸链复合体III的活性，并表现出明显的剂量和时间依赖性；荧光实时定量PCR分析表明，天明精内酯酮EC50剂量处理菌丝体后，编码线粒体呼吸链复合体Ⅲ的亚基基因GgCyc1表达量明显下调，而与复合体Ⅰ、Ⅱ、Ⅳ和Ⅴ相关的GgNus, GgSdcb等5个亚基基因表达量均上调。.综上可见，天名精内酯酮主要作用于病菌线粒体，其靶标可能是线粒体呼吸链复合体III，可能的抑菌机理为：天名精内酯酮抑制复合体III的活性，阻断电子传导，而菌体CAT、SOD和GSH-PX活性的变化，进一步导致病菌线粒体活性氧大量积累，从而激活线粒体介导的细胞凋亡，最终导致菌体死亡。但靶标的确定尚欠缺直接证据，需进一步研究。.项目结果为进一步揭示天名精内酯酮的抑菌分子机理，促进其产业化开发奠定了良好基础。