鸦胆子素D靶向Cathepsin B诱导斜纹夜蛾幼虫中肠细胞凋亡的分子机制

鸦胆子素D（BD）是申请人从鸦胆子药渣中分离得到的兼具高效杀虫与内吸活性的化合物。前期发现，BD显著上调斜纹夜蛾幼虫中肠Cathepsin B表达，损伤中肠组织，抑制幼虫生长发育，暗示Cathepsin B是BD诱导斜纹夜蛾中肠细胞凋亡致其损伤的靶基因。为证明并解析其作用机理，拟在虫体水平动态追踪BD对中肠细胞凋亡的影响与代谢功能损伤，运用共聚焦显微镜等手段在SL-1细胞中可视化观察Cathepsin B被诱导上调并从溶酶体释放至细胞质破坏线粒体的过程；通过透射电镜、Western blot等技术在细胞及虫体水平揭示BD诱导细胞凋亡的通路；利用RNAi和杆状病毒技术构建Cathepsin B低/高表达虫体，研究其对BD的营养效应与中肠的病理变化，最终阐明Cathepsin B在BD诱导斜纹夜蛾中肠凋亡致其损伤中的分子功能,为鸦胆子素D杀虫剂的开发提供理论基础。

Bruceine D was isolated from the herb residue of Brucea javanica (L.), displayed high insecticidal and systemic activity. It was found that bruceine D can significantly up-regulate the expression of cathepsin B in the midgut of Spodoptera litura larvae, and damage the larval midgut tissue, thereby inhibiting the growth and development of the larva, suggesting that Cathepsin B was the target gene for bruceine D to induce apoptosis in the midgut cell of Spodoptera litura. In order to uncover its mechanism of action, We firstly intends to dynamically track the effect of bruceine D inducing midgut cell apoptosis and metabolic function damage in vivo, and visualize the cathepsin B up-regulated and released from the lysosome to the cytoplasm to destroy the mitochondrial structure at cellular level by confocal microscopy; then, to reveal the pathway of bruceine D-induced apoptosis at the cell and larval level through western blot and transmission electron microscopy technique; the strains of Spodoptera litura with RNAi or over expression of cathepsin B of will be constructed using RNAi and bac-to-bac expression systems, and their nutritional effect on Bruceine D and the pathological changes of the midgut will be studied. Finally, Molecular function of cathepsin B involved in the damage of the midgut of Spodoptera litura induced by Bruceine D will be elucidated. This project provides a theoretical basis for the commercialization of bruceine D insecticide.