雷公藤次碱作用于粘虫肌细胞的靶标定位研究

Alkaloids are primary insecticidal compounds isolated from Tripterygium wilfordii Hook.f. Our preliminary studies showed that muscle tissues of Mythimna separate were significantly damaged by Tripterygium wilfordii Hook.f. alkaloids (ATW), indicating that ATW’s target should be located in muscle cells. In this project, with Wilforine, the highest insecticidal activity component of ATW as the research chemical, larvae and adults of Mythimna separate as well as flight muscle cells of the adult insect as tested materials, there are three main missions: 1) according to the effects on morphology and function of the muscle cells exposed by wilforine, expressions of proteins relevant to Ca2+ regulation is planned to determine through RT-PCR, 2) with the prepared monoclonal antibody, the subcellular target locations are aims to elucidated by means of immune-colloidal gold technology, 3) through laser scanning confocal microscope combined with Ca2+ fluorescence indicator, dynamic alterations of free Ca2+ in the muscle cells in vitro are scheduled to determine to identify the effects on Ca2+ channel and to elucidate its mechanisms as well as targets. The objectives of this research are to lay a foundation for elucidating insecticidal mechanisms of ATW and to provide theory basements for new insecticides with insect muscle cells as their targets.

雷公藤生物碱是杀虫植物雷公藤（Tripterygium wilfordii Hook. f.）的主要活性成分，前期研究表明该类化合物可造成昆虫肌肉组织病变，作用靶标可能存在于肌细胞。本项目拟以雷公藤生物碱中杀虫活性最强的雷公藤次碱为研究对象，以粘虫为供试昆虫，对其作用靶标进行定位研究，具体为：在明确其对肌细胞形态与活力影响的基础上，分析其对离体肌细胞Ca2+调控相关蛋白表达的影响，探讨其对肌细胞的毒性；通过制备单克隆抗体，以免疫胶体金技术对其在肌细胞上靶标位点进行亚细胞定位；进一步采用激光共聚焦显微镜技术结合Ca2+荧光指示剂，测定离体肌细胞胞浆中游离[Ca2+]i动力学变化，分析其对钙通道的作用，进行分子靶标定位，并探讨可能的作用机理。该研究旨在逐级准确定位雷公藤次碱的作用靶标，为阐明植物源杀虫活性物质雷公藤次碱的杀虫机理奠定基础，也为一类以昆虫肌细胞为靶标新型杀虫剂的创制提供依据。

探究天然产物的分子靶标，历来是农药科学领域的研究热点和前沿。具我国自主知识产权的雷公藤生物碱杀虫剂因作用机理不清而限制了产品开发及应用。前期研究表明该类化合物可造成昆虫肌肉组织病变，作用靶标可能存在于肌细胞。为探究该类化合物的杀虫作用机理，项目以雷公藤生物碱中杀虫活性最强的雷公藤次碱（WR）为研究对象，以东方粘虫为供试昆虫，以氯虫苯甲酰胺（CT）为对照药剂，对其作用靶标进行了定位研究。主要结果为：组织病理学观察显示，WR可致粘虫肌肉组织结构病变，对肌细胞膜及胞内细胞器（肌质网等）均具有破坏作用，且病变程度随中毒症状的加深而加重；肌细胞钙动力学测定表明，WR可作用于鱼尼丁受体（RyR），引起肌质网钙库中的钙离子释放；转录组测序结合qRT-PCR验证发现，WR对RyR及Actin等与肌肉收缩/舒张相关蛋白基因的表达量均有明显影响，其中，WR处理可致RyR基因表达量降低，CT处理可致RyR基因表达量升高，且均具有时间和剂量依赖性；RNAi表明，注射dsMsRyR后，试虫对WR和CT的敏感性明显下降。对照药剂CT在致毒症状、相关基因表达、钙动力学变化等方面与WR有一定的相似性和明显的差异性。可见，WR的分子靶标位于昆虫的肌细胞，RyR为其疑似靶标。结合症状学及毒理学研究结果，推测WR作用于RyR使得肌细胞内 Ca2+浓度失衡，引起昆虫正常的肌肉收缩/舒张紊乱，昆虫瘫软、麻痹，最终死亡。项目结果在揭示WR杀虫机理中取得了突破性进展，初步明确了WR的杀虫机理，且提出RyR为其疑似靶标的论断，为进一步探究该类化合物的杀虫分子靶标奠定了坚实的基础。