小菜蛾钙黏蛋白PxCR_(10-11)结构域的克隆、原核表达及功能分析

Cloning,Prokaryotic Expression and Functional Analysis of PxCR_(10-11) Domain in Plutella xylostella Cadherin

钙黏蛋白(cadherin)是一类跨膜糖蛋白,因其在苏云金芽胞杆菌(Bacillus thuringiensis,Bt)的杀虫过程中作为主要的受体而受到广泛研究.钙黏蛋白的特征之一是由若干钙黏蛋白重复单元(cadherin repeats,CR)组成的长链状蛋白,其中靠近细胞膜的CR结构域被认为是钙黏蛋白与Bt毒素发生互作的区域.小菜蛾(Plutella xylostella)钙黏蛋白由11个CR结构域和1个跨膜结构域组成,其中第10和11个CR结构域PxCR_(10-11)被认为是钙黏蛋白与Bt毒素的互作区域.本研究从小菜蛾中肠cDNA中克隆得到小菜蛾钙黏蛋白PxCR_(10-11)结构域的DNA片段,并通过原核表达系统对PxCR_(10-11)结构域进行表达.配体印迹结果表明PxCR_(10-11)可以特异性和Cry2Ab结合;杀虫实验结果表明PxCR_(10-11)能提高Cry2Ab对小菜蛾的毒力.此外,利用蛋白质同源建模和糖基化位点预测网站对PxCR_(10-11)蛋白质结构进行了分析.上述结果表明PxCR_(10-11)可能参与了Cry2Ab毒素对小菜蛾的毒杀过程,为后续研究小菜蛾钙黏蛋白和Cry2Ab的相互作用机制奠定了基础.

Cadherin,formed by a number of characteristic cadherin repeats（CR）domains,is a chain-like membrane glycoprotein.It was widely reported that cadherin in insect midgut played an essential role in the insecticidal activitives of Bacillus thuringiensis（Bt）toxins.The CR domain near the cell membrane was documented as an interaction region with Bt toxins.Plutella xylostella cadherin PxCR was formed by 11 CR domains and a transmembrane domain.The 10th and 11th CR domains（PxCR10-11）in PxCR were regarded as a potential interaction region with Bt toxins.In this study,PxCR10-11 domain was cloned from Plutella xylostella midgut cDNA,and then a prokaryotic expression vector pGEX4t-PxCR10-11 was constructed and the fusion protein GST-PxCR10-11 was induced to express.Ligand blot indicated that PxCR10-11 could specially bind to Cry2Ab and the bioassay revealed that PxCR10-11 had synergistic effect on Cry2Ab against Plutella xylostella larvae.Furthermore,homologous modeling of PxCR10-11 was conducted and the glycosylation sites in PxCR10-11 were predicted.These results indicate that PxCR10-11 is involved in the processing of Cry2Ab against Plutella xylostella larvae,which lays the foundation for the interaction mechanism of PxCR10-11 with Cry2Ab toxins.