苏云金芽胞杆菌Cry1Ba3 蛋白定点突变对杀小菜蛾活性的影响

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Ple 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。

The influences of site directed mutation on the toxicity of Bacillus thuringiensis Cry1Ba3 polypeptide against Plutella xylostella

Cry1Ba3 is an insecticidal crystal protein revealed by our laboratory. It has high toxicity against Plutella xylostella. The object of this research is to find amino acids that are important to its insecticidal activity and to provide a theoretical basis for the research on pesticidal mechanism and recombination of insecticidal proteins. Firstly, the database Ple Bio Informatique Lyonnais was used to predict the secondary structure of Cry1Ba3; secondly, hydrophobic regions were found after analyzing the protein sequence of Cry1Ba3 with the software called BioEdit; finally, 20 amino acid residue sites were determined after multiple alignments between Cry1Ba3, Cry1Aa1, Cry2Aa1, Cry3Aa1 and Cry4Ba1. Over lapping PCR was used to make site directed mutation on Cry1Ba. Twenty mutants were expressed in Escherichia coli BL21 (DE3) strain respectively, and the bioassays against Plutella xylostella were conducted with the proteins expressed in the mutants. The results showed that 20 mutant proteins could be expressed normally with a molecular weight of 66 ku in BL21 strain and existed as inclusion bodies. Bioassay results showed that the toxicities of mutant M6 (R192L), M10 (W303A) and M19 (H485G) were decreased significantly. The prediction results of secondary structure of the mutants indicated that the toxin conformation of the mutants whose toxicity decreased sharply was changed markedly. However, neither the toxicity nor the secondary structure of the other 17 mutants was changed obviously. Arginine on site 192, tryptophan on site 303 and histidine on site 485 were very important for the toxicity of Cry1Ba3 against P. xylostella, and the reduction of toxicity was related to the diversification of toxin conformation caused by the mutations of the three amino acid residues.