Synergism of Bacillus thuringiensis Toxin Cry1Ac by a Fragment of Toxin-Binding Polycalin from Plutella xylostella.
Synergism of Bacillus thuringiensis Toxin Cry1Ac by a Fragment of Toxin-Binding Polycalin from Plutella xylostella.
复制标题
DOI：
10.1021/acs.jafc.1c03156
复制
发表时间：
2021-10
影响因子：
6.1
通讯作者：
Qing Zhu;M. Gao;Lina Lu;Xianjin Liu
中科院分区：
文献类型：
--
作者：
Qing Zhu;M. Gao;Lina Lu;Xianjin Liu
利用苏云金芽孢杆菌(Bt)杀虫晶体蛋白(Cry)在转基因植物中控制害虫的持续成功受到抗性进化的威胁。以往的研究表明，小菜蛾多钙蛋白可以作为一种潜在的受体与Cry1Ac毒素结合。本研究克隆并表达了小菜蛾多钙蛋白(PxPolycalinf，G2209-A2942)的一个含羧基末端GPI锚定信号肽的片段。在生物测定中，纯化的PxPolycalinf保留了与Cry1Ac结合的能力，并增强了Cry1Ac对小菜蛾三龄幼虫的毒性。此外，PxPolycalinf的多克隆抗体与正常食物一起喂养后，降低了Cry1Ac的活性。进一步的ELISA法结果表明，PxPolycalinf与小菜蛾刷状缘膜小泡(BBMV)的结合呈浓度依赖性。表达PxPolycalinf的果蝇Sf9细胞对Cry1Ac不敏感，而表达PxABCC2的果蝇Sf9细胞对Cry1Ac的细胞毒作用增强。免疫定位显示PxPolycalinf与Sf9细胞膜结合，ELISA显示PxPolycalinf与Sf9细胞提取液结合呈浓度依赖关系。这些结果首次证明了Cry1Ac的潜在受体--小菜蛾多钙蛋白片段对小菜蛾幼虫和表达PxABCC2的Sf9细胞具有协同作用。
The continued success of pest control using insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) in transgenic plants was threatened by the evolution of resistance. Previous studies suggested that polycalin from Plutella xylostella could bind to Cry1Ac toxin as a potential receptor. In this study, a fragment of P. xylostella polycalin (Pxpolycalinf, G2209-A2942) containing a carboxyl-terminal GPI-anchored signal peptide was cloned and expressed. Purified Pxpolycalinf retained the binding ability to Cry1Ac and synergized Cry1Ac toxicity to the third larvae of P. xylostella in bioassays. Moreover, the polyclonal antibody of Pxpolycalinf decreased the Cry1Ac activity after being fed together with normal food. Further, the ELISA results showed the concentration-dependent binding of Pxpolycalinf to P. xylostella brush border membrane vesicles (BBMV). Spodoptera frugiperda 9 (Sf9) cells expressing Pxpolycalinf were not susceptive to Cry1Ac, whereas Pxpolycalinf increased Cry1Ac cytotoxicity to Sf9 cells expressing P. xylostella ATP-dependent binding cassette transporter C2 (PxABCC2). Immunolocalization presented the binding of Pxpolycalinf to the Sf9 cell membrane, and ELISA showed the concentration-dependent binding of Pxpolycalinf to Sf9 cell extraction. These results here provide the first evidence that a fragment of P. xylostella polycalin, a potential receptor of Cry1Ac, synergizes Cry1Ac toxicity to P. xylostella larvae and Sf9 cells expressing PxABCC2.