家蚕氨肽酶和类钙粘蛋白与苏云金芽孢杆菌Cry1Ac毒素相互作用

Interaction of Bombyx mori aminopeptidase N and cadherin-like protein with Bacillus thuringiensis Cry1Ac toxin

摘要:

苏云金芽孢杆菌Bacillus thuringiensis生产的晶体毒素被广泛用作农林害虫的杀虫剂。鳞翅目昆虫受体蛋白是阐明其与晶体毒素相互作用的重要模式。文中纯化了苏云金芽孢杆菌的晶体毒素蛋白，质谱鉴定为Cry1Ac毒素，然后重组表达家蚕氨肽酶N (BmAPN6) 和类钙粘蛋白 (CaLP) 结合结构域。利用免疫共沉淀、Far-Western印迹和酶联免疫吸附试验，证明Cry1Ac毒素蛋白和BmAPN6之间的相互作用。在Sf9细胞中，对Cry1Ac毒素的细胞毒活性分析，表明BmAPN6参与Cry1Ac毒素诱导的细胞形态异常和裂解死亡。文中也利用相同的方法，对钙粘蛋白的3个结合位点CR7、CR11和CR12进行相互作用分析，结果表明3个重复结构域是CaLP的Cry1Ac结合位点。上述结果表明，BmAPN6和CaLP可作为Cry1Ac毒素致病的功能性受体，为进一步揭示晶体毒素的致病机制和基因编辑增强家蚕抗病性提供了研究靶标。

Abstract:

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.