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Cry1Ba3是由本实验室发掘的对小菜蛾有高毒力的杀虫晶体蛋白。为了寻找对杀虫活性有重要影响的氨基酸,为杀虫机理研究和改造杀虫蛋白提供理论依据,本研究利用Ple Bio-Informatique Lyonnais数据库对Cry1Ba3的二级结构进行模拟;采用BioEdit软件分析Cry1Ba3的疏水区;将Cry1Ba3与Cry1Aa1、Cry2Aa1、Cry3Aa1以及Cry4Ba1进行多序列比对,从而确定了20个氨基酸突变位点。利用半重叠引物PCR的方法对Cry1Ba3进行定点突变,将突变体在大肠杆菌BL21中进行诱导表达。通过浸叶法对各个突变蛋白杀小菜蛾的生物活性进行测定。获得的20个Cry1Ba3突变体均能在大肠杆菌BL21中表达,并以包涵体形式存在。杀虫活性测定结果表明M6(R192L)、M10(W303A)、M19(H485G)对小菜蛾的活性明显降低,二级结构预测表明活性降低的突变蛋白的构象均发生明显变化,其余17种突变蛋白的活性和二级结构都没有明显变化。说明第192位的精氨酸、第303位的色氨酸和第485位的组氨酸对Cry1Ba3的杀小菜蛾活性有重要影响,上述3个位点氨基酸突变引起的蛋白活性减低可能与毒素的空间结构变化有关。
Cry1Ba3 is an insecticidal crystal protein that is highly virulent to diamondback moth in our laboratory. In order to find the amino acids that have an important influence on insecticidal activity and provide a theoretical basis for the insecticidal mechanism research and the transformation of insecticidal proteins, the secondary structure of Cry1Ba3 was simulated by Ple Bio-Informatique Lyonnais database. BioEdit software was used to analyze Cry1Ba3 hydrophobic region; Cry1Ba3 with Cry1Aa1, Cry2Aa1, Cry3Aa1 and Cry4Ba1 multiple sequence alignment to determine the 20 amino acid mutation sites. Cry1Ba3 was subjected to site-directed mutagenesis using semi-overlapping primer PCR, and the mutant was induced in E. coli BL21. The biological activity of each mutant protein against Plutella xylostella was determined by dipping leaf method. All of the 20 Cry1Ba3 mutants obtained were expressed in E. coli BL21 and existed as inclusion bodies. The results of insecticidal activity showed that the activities of M6 (R192L), M10 (W303A) and M19 (H485G) on Plutella xylostella were significantly reduced, and the secondary structure prediction indicated that the conformation of the mutein with reduced activity changed significantly. The remaining 17 muteins The activity and secondary structure did not change significantly. This indicated that arginine at position 192, tryptophan at position 303 and histidine at position 485 had an important effect on the activity of CrylBa3 against Plutella xylostella. The reduction in protein activity caused by amino acid mutation at the above three positions may be related to that of toxin The spatial structure of the changes.