苏云金芽胞杆菌（Bacillus thuringiensis，Bt）产生的营养期杀虫蛋白（Vegetative insecticidal proteins，VIPs）Vip3类对鳞翅目害虫具有良好的杀虫活性。Vip3蛋白与Cry蛋白氨基酸序列相似性极低且杀虫作用机制不同，因此，在转基因作物上叠加使用vip3与cry基因可以延缓靶标昆虫抗性的产生。为了获得对鳞翅目害虫具有高毒力的新型Vip3类杀虫蛋白，本研究以实验室前期克隆的vip3Aa基因为模板利用定点突变技术构建突变体，原核表达后的突变体蛋白对鳞翅目害虫进行杀虫活性的测定和杀虫机制的分析，研究结果如下：

（1）基于已报道的Vip3Aa突变体蛋白对甜菜夜蛾杀虫活性的研究，参考其6个关键氨基酸位点，并在此基础上，构建了4个多氨基酸位点突变体，这些突变体在大肠杆菌中均表达为可溶性蛋白。杀虫活性测定结果表明，Vip3Aa蛋白对草地贪夜蛾初孵幼虫的LC₅₀为0.946 μg/g，Vip3Aa-S543N/I544L/E627A和S543N/I544L/S686R突变体蛋白的LC₅₀分别为0.118 μg/g和0.365 μg/g，杀虫活性分别提高8倍和2.6倍；Vip3Aa蛋白对棉铃虫初孵幼虫的LC₅₀为11.528 μg/g，Vip3Aa-S543N/I544L/S686R突变体蛋白的LC₅₀为3.577 μg/g，杀虫活性提高3.2倍；在浓度为100 µg/g时，Vip3Aa蛋白对草地贪夜蛾的校正死亡率为93.8%，而Vip3Aa-W552A和N624A突变体蛋白的校正死亡率仅为2.1%，丧失了对草地贪夜蛾的杀虫活性。

（2）中肠液体外活化实验结果表明，Vip3Aa-S543N/I544L/E627A和S543N/I544L/S686R突变体蛋白在草地贪夜蛾和棉铃虫的中肠液中比Vip3Aa蛋白更稳定，Vip3Aa-N624A突变体蛋白被草地贪夜蛾中肠液完全活化。

（3）通过酶联免疫测定法（ELISA）检测Vip3Aa和突变体蛋白与鳞翅目害虫中肠BBMVs之间的结合能力，结果表明，Vip3Aa蛋白与草地贪夜蛾BBMVs结合的解离常数Kd=87.92±13.03nmol/L，Vip3Aa-N624A突变体蛋白的Kd=195.78±18.89 nmol/L，Vip3Aa-S543N/I544L/E627A突变体蛋白的Kd=17.324±4.14 nmol/L，Vip3Aa-S543N/I544L/S686R突变体蛋白的Kd=33.83±7.22 nmol/L，与Vip3Aa蛋白相比，Vip3Aa-S543N/I544L/E627A和S543N/I544L/S686R突变体蛋白与草地贪夜蛾BBMVs结合能力分别提高5倍和2.6倍，Vip3Aa-N624A突变体蛋白与草地贪夜蛾BBMVs结合能力降低2.2倍；Vip3Aa蛋白与棉铃虫BBMV  s结合的解离常数Kd=224.77±30.28 nmol/L，Vip3Aa-S543N/I544L/S686R突变体蛋白的Kd=69.70±8.78 nmol/L，与Vip3Aa蛋白相比，Vip3Aa-S543N/I544L/S686R突变体蛋白与棉铃虫BBMVs结合能力提高3.2倍。

（4）Vip3蛋白的第Ⅳ和第Ⅴ结构域为非保守区域，这个区域与杀虫特异性有关，其中loop区域很有可能参与受体的结合，因此，选取了暴露在外侧的loop区域氨基酸位点进行突变，成功获得10个可溶性突变蛋白。杀虫活性测定结果表明，Vip3Aa蛋白对草地贪夜蛾初孵幼虫的LC₅₀为1.267 μg/g，Vip3Aa-K588A/K590A/K592A、S689A和N773A/N774A突变体蛋白的LC₅₀分别为6.600 μg/g、6.936 μg/g和4.558 μg/g，杀虫活性分别降低5.2倍、5.5倍和3.6倍；Vip3Aa-D728A、L775A/Y776A/G777A和G778A/P779A/I780A突变体蛋白在浓度为200 μg/g时，对草地贪夜蛾未表现出明显的杀虫活性。

本研究获得了较Vip3Aa蛋白杀虫活性显著提高的突变体蛋白，为获得高毒力蛋白提供思路，为鳞翅目害虫的防治提供新的资源；同时，分析了杀虫活性产生差异的原因并探索了蛋白质氨基酸对杀虫活性的影响，为Vip3Aa蛋白杀虫机理的研究奠定基础。

Vegetative insecticidal proteins (VIPs) produced by Bacillus thuringiensis (Bt) have good insecticidal activity against lepidopteran pests. Vip3 proteins do not share homology in sequence with Cry proteins and their mechanism of action is different. Vip3Aa has no cross resistance with Cry proteins, therefore, vip3 and cry genes are often co-expressed in transgenic crops to delay development of insect resistance. In order to obtain novel Vip3 insecticidal proteins with higher toxicity against lepidopteran insects, the vip3Aa gene cloned in our laboratory was used as material to construct mutants by site-directed mutagenesis. The insecticidal activity of the expressed proteins against lepidopteran pests and and analysis of insecticidal mechanism were determined. The results are as follows:

(1) Based on the reported insecticidal activity of Vip3Aa mutant protein against S. exigua, four mutants with multiple amino acid sites were constructed according to its six key amino acid sites. These mutants were expressed as soluble proteins in E. coli. The bioassay results showed that the LC₅₀ of wild type protein against S. frugiperda was 0.946 μg/g. The LC₅₀ of Vip3Aa-S543N/I544L/E627A and S543N/I544L/S686R mutant protein against S. frugiperda were 0.118 μg/g and 0.365 μg/g, respectively, and the insecticidal activity increased 8-fold and 2.6-fold. The LC₅₀ of wild-type protein against H. armigera was 11.528 μg/g, and that of Vip3aA-S543N /I544L/S686R mutant protein was 3.577 μg/g, the insecticidal activity increased 3.2-fold. At the concentration of 100 µg/g, the corrected mortality of wilt-type protein against Spodoptera frugiperda was 93.8% and the corrected mortality of Vip3Aa-W552A and N624A mutant protein were only 2.1%, indicating that the two mutant proteins lost their insecticidal activity against S. frugiperda.

(2) Experimental results of intestinal fluid activation in vitro, Vip3Aa-S543N/I544L/E627A and S543N/I544L/S686R mutant proteins were more stable than wild-type proteins in the midgut fluid of S. frugiperda and H. armigera. Vip3Aa-N624A mutant protein was completely activated by the midgut fluid of S. frugiperda.

(3) The results of ELISA showed that the dissociation constant of wild-type protein with BBMVs of S. frugiperda was 87.92±13.03 nmol/L, and the Kd of Vip3Aa-N624A mutant protein was 195.78±18.89 nmol/L. The Kd of Vip3Aa-S543N/I544L/E627A and Vip3aA-S543N/I544L/S686R mutant protein were 17.324±4.14 nmol/L and 33.83±7.22 nmol/L, respectively. The binding ability of Vip3aA-S543N/I544L/E627A and S543N/I544L/S686R mutant protein to BBMVs of S. frugiperda were increased 5-fold and 2.6-fold, respectively, while the binding ability of Vip3aA-N624A protein was decreased 2.2-fold. The dissociation constant of Vip3Aa protein to BBMVs of H. armigera was 224.77±30.28 nmol/L, and the Kd of Vip3Aa-S543N/I544L/S686R mutant protein was 69.70±8.78 nmol/L. The binding ability of Vip3aA-S543N/I544L/S686R mutant protein to BBMVs of H. armigera was increased 3.2-fold.

(4) The IV and V domains of Vip3 protein are non-conservative regions, which are related to insecticidal specificity. The loop region is likely to participate in the binding of receptors. Therefore, the amino acid sites of the loop region exposed to the outside were selected for mutation, and 10 soluble mutant proteins were successfully obtained. The bioassay results showed that the LC₅₀ of wild type protein against S. frugiperda was 1.267 μg/g. The LC₅₀ of Vip3Aa-K588A/K590A/K592A, S689A and N773A/N774A mutant proteins were 6.600 μg/g, 6.936 μg/g and 4.558 μg/g, respectively, and the insecticidal activity decreased 5.2-fold, 5.5-fold and 3.6-fold. Vip3Aa-D728A, L775A/Y776A/G777A and G778A/P779A/I780A mutant proteins showed no significant insecticidal activity against S. frugiperda at the concentration of 200 μg/g.

In this study, several mutant proteins with higher insecticidal activity than the wild-type protein were obtained, which provided new ideas for obtaining highly virulent proteins and provided new resources for the control of lepidopteran pests. At the same time, the reasons for the changes of insecticidal activity were analyzed. The exploration of key amino acid sites for insecticidal activity lay a foundation for the study of insecticidal mechanism of Vip3Aa protein.

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