以啶虫脒为生物偶联配基的Bt毒素定向改造和结构表征

Improving the affinity between Bt toxin and its receptor proteins is key to overcome Bt toxin resistance, which is also the main approach for directed engineering of Bt toxin. In our previous studies, acetamiprid (Ac) was screened as a lead compound for the bioconjugate ligand of Cry2Ab by bioinformatics from chemical pesticides. Acetamiprid mercaptopropionic acid was then synthesized by nucleophilic substitution and coupled with Cry2Ab. This engineering of Cry2Ab improved the affinity between Cry2Ab and its receptor proteins on the BBMV of Plutella xylostella. To further investigate the effect of the length of mercaptan acid to the affinity between Cry2Ab and its receptor protein (PxAPN5) on the BBMV of Plutella xylostella, a series of acetamiprid mercaptan acids were synthesized and coupled with Cry2Ab to form novel toxins Cry2Ab-Ac. The affinity between Cry2Ab-Ac and PxAPN5 was measured by protein-protein interaction system (Octet red). The activity of Cry2Ab-Ac to the Bt-resistant of Plutella xylostella was then analyzed. The advanced characterization of Cry2Ab-Ac was performed by LC-MS, LC-MS/MS, HIC-UV and so on. This directed engineering of Bt insecticidal protein may provide a new platform for the development of biological insecticide.

提高Bt毒素与受体的互作，是治理害虫抗性的关键，也是Bt毒素定向改造的方向。申请者团队在前期啶虫脒巯基丙酸的结构改造及其与Bt Cry2Ab毒素偶联修饰研究的基础上，探索不同碳链长度的啶虫脒巯基酸的偶联修饰对Cry2Ab活性的影响。合成不同碳链长度的系列啶虫脒巯基酸（啶虫脒巯基乙酸、丙酸和丁酸等）作为Cry2Ab的偶联配基对Cry2Ab定向改造，合成偶联型毒素Cry2Ab-Ac，以期提高Cry2Ab与小菜蛾中肠受体氨肽酶的互作，并探索Cry2Ab-Ac对小菜蛾抗性治理的效果。利用偶联药物鉴定分析技术鉴定偶联型毒素Cry2Ab-Ac，测定偶联比，鉴定偶联位点，对Cry2Ab-Ac的结构进行表征。此工作将为Bt毒素的定向改造及新型生物农药的研发奠定理论基础。

新型Bt农药的研发是农业绿色生产的前沿问题。提高Bt杀虫毒素与昆虫受体蛋白的互作，是提高Bt农药防效的关键机制。项目利用生物偶联技术，筛选系列啶虫脒巯基酸为偶联配基，在偶联剂EDC+NHS的作用下，将其与Cry2Ab蛋白共价偶联，合成系列偶联型Cry2Ab-Ac。利用SDS-PAGE、高分辨质谱和肽指纹图谱等技术，鉴定偶联结果，表征偶联型Cry2Ab-Ac的结构，测定偶联位置。利用Octet Red蛋白质互作系统研究偶联修饰前后Cry2Ab与其在小菜蛾中肠受体蛋白PxAPN5的互作，发现偶联修饰能极显著地提高Cry2Ab与受体PxAPN5的结合力，其中偶联配基啶虫脒巯基丙酸（Ac-MPA）改造的效果最佳，解离常数（KD）仅为Cry2Ab的16%。这与偶联修饰前后Cry2Ab对小菜蛾的杀虫毒力的结果相一致。偶联修饰显著提高Cry2Ab对小菜蛾的杀虫毒力，啶虫脒巯基丙酸修饰的效果最佳，修饰后的Cry2Ab对小菜蛾的杀虫毒力是修饰前的119倍。项目还进行了拓展研究，分析空白饲料，Cry2Ab和Cry2Ab-Ac-MPA饲喂小菜蛾后转录组表达的差异。共鉴定出9848个差异表达基因，在“protein metabolic process”，“cellular protein metabolic process”，“cytoplasm”和“oxidoreductase activity”等功能和“glutathione metabolism”、“protein processing in endoplasmic reticulum”、“oxidative phosphorylation”和“metabolism of xenobiotics by cytochrome P450”等通路差异较为显著。偶联修饰后的Cry2Ab-Ac-MPA能激发小菜蛾更强的免疫应答，在信号因子的识别及结合等都优于Cry2Ab。q-PCR验证偶联修饰前后Cry2Ab作用小菜蛾后的差异表达基因的结果也显示偶联修饰后的Cry2Ab-Ac-MPA饲喂小菜蛾后NOG1、rL12、CHP1和Protein rolling stone等基因显著上调。这为偶联配基的筛选及新型Bt农药的研发和应用推广，提供理论支撑。