苏云金芽孢杆菌杀虫蛋白cry2Ad2原核表达及其不同长度片段产物的杀虫活性

Prokaryotic expression and toxicity analysis of truncated insecticidal protein Cry2Ad2 from Bacillus thuringiensis

该研究采用NCBI蛋白分析软件对Cry2Ad2蛋白序列进行分析,采用PCR克隆获得3种不同长度的cry2Ad2及全长cry2Ad2基因片段,并将这些基因片段克隆到pET-26b载体上,构建重组质粒pET-Ad、pET-Ad1、pET-Ad2和pET-Ad3,再导入大肠杆菌BL21(DE3)中进行细胞周质诱导表达,并采用室内生物测定法测定表达产物对小菜蛾的毒力。Cry2Ad2蛋白的序列分析结果表明:全长的Cry2Ad2蛋白包含两个保守区域domain I(第53位～第264位氨基酸)和domainⅡ(第267位～第472位氨基酸),推测保守活性片段C端末端在N-端的第472位与第633位氨基酸之间。重组质粒pET-Ad、pET-Ad1、pET-Ad2和pET-Ad3在大肠杆菌BL21(DE3)中,经IPTG诱导表达,SDS-PAGE电泳验证分别含有约70 000、52 000、51 000和54 000的表达蛋白。生物活性初步测定结果表明:表达的Cry2Ad2、Cry2Ad2-1、Cry2Ad2-2及Cry2Ad2-3给药72 h后对小菜蛾都具有杀虫活性,其中蛋白Cry2Ad2、Cry2Ad2-2与Cry2Ad2-3对小菜蛾有较强的毒性,相同剂量致死率分别为64.35%、53.27%和61.66%,统计分析表明蛋白Cry2Ad2-1与Cry2Ad2-3的致死率与蛋白Cry2Ad2的致死率无显著差异,(P>0.05);蛋白Cry2Ad2-2对小菜蛾的毒力相对较低,相同剂量致死率为39.89%,蛋白Cry2Ad2-2的致死率与蛋白Cry2Ad2的致死率差异显著(P<0.05)。鉴此,初步推断Cry2Ad2蛋白对小菜蛾的活性区在第49位与第463位氨基酸之间。

A putative full-length amino acid sequence of cry2Ad2 was functionally annotated using NCBI′s conserved domain database（CDD） to identify the conserved domains and provide insights into phylogenetic relationships.According to the annotation results,a full-length and three truncated fragments of cry2Ad2 was isolated and expressed in Escherichia coli BL21.The Cry2Ad2 protein contains two conserved domains,domain I（amino acids 53 to 264） and domain Ⅱ（amino acids 267 to 472）,implying that the minimum functional unit of Cry2Ad2 ranged from amino acids 53 to 472.Four polypeptide bands,corresponding to molecular masses of about 70 000（Cry2Ad2）,52 000（Cry2Ad2-1）,51 000（Cry2Ad2-2） and 54 000（Cry2Ad2-3） were identified by SDS-PAGE.All of the expressed proteins revealed toxic activity to Plutella xylostella.Cry2Ad2（amino acids 1 to 633）,Cry2Ad2-1（amino acids 4 to 463） and Cry2Ad2-3（amino acids 49 to 491） showed high insecticidal activity of the assayed pests at the same treated concentration,while the Cry2Ad2-2（amino acids 65 to 633） has lower in sedicidal activity with 39.89% mortality.This result also indicated that the minimum functional unit of protein Cry2Ad2 could be limited to amino acids 49 to 463.