Study On Vip3Aa Mutant With High Virulence To Spodoptera Frugiperda

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 0.946μg/g.The LC₅₀ of Vip3Aa-S543N/I544L/E627A and S543N/I544L/S686R mutant protein against S.frugiperda were 0.118μg/g and 0.365μg/g,respectively,and the insecticidal activity increased 8-fold and 2.6-fold.The LC₅₀ of wild-type protein against H.armigera was 11.528μg/g,and that of Vip3a A-S543N/I544L/S686R mutant protein was 3.577μg/g,the insecticidal activity increased 3.2-fold.At the concentration of 100μg/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±18.89nmol/L.The Kd of Vip3Aa-S543N/I544L/E627A and Vip3a A-S543N/I544L/S686R mutant protein were17.324±4.14 nmol/L and 33.83±7.22 nmol/L,respectively.The binding ability of Vip3a A-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 Vip3a A-N624A protein was decreased 2.2-fold.The dissociation constant of Vip3Aa protein to BBMVs of H.armigera was224.77±30.28 nmol/L,and the Kd of Vip3Aa-S543N/I544L/S686R mutant protein was 69.70±8.78nmol/L.The binding ability of Vip3a A-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 1.267μg/g.The LC₅₀ of Vip3Aa-K588A/K590A/K592A,S689A and N773A/N774A mutant proteins were 6.600μg/g,6.936μg/g and 4.558μg/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 200μg/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.