Directed Modification Of Cry2ab Protein With High-efficiency Insecticidal Activity Against Spodoptera Frugiperda

Spodoptera frugiperda is an agricultural pest that is native to tropical and subtropical America.It has a strong ability to migrate.Since January 2019,Spodoptera frugiperda has invaded China and spread rapidly throughout the country.This poses a major threat to China’s food production security and ecological security.At present,chemical control and biological control are the two main control strategies to Spodoptera frugiperda.Biological control mainly relies on insect pathogens such as Bacillus thuringiensis（Bt）.The latest research shows that Bt Cry2Ab protein has certain insecticidal activity against Spodoptera frugiperda in China.This research focuses on the cry2A genes and the Cry2Ab29 mutants on insecticidal activity against Spodoptera frugiperda.The difference of insecticidal activity of Cry2A protein and the key amino acid sites affecting insecticidal activity were analyzed.The main results are as follows:1.The insecticidal activity of Cry2A proteins against Spodoptera frugiperda were investigated.In order to solve the problem of poor solubility of Cry2A proteins in E.coli expression system,six cry2A genes were successfully expressed in Bt.Under the light microscope,only Cry2Aa9 and Cry2Ab4 can be observed square crystals.The crystals of the other four strains were irregular.In order to further clarify the specific morphology of the two crystals,the Cry2Ab4 and Cry2Ab29 crystal were observed by atomic force microscopy（AFM）.The solubility of Cry2A crystal protein was analyzed by five different lysates.The results showed that Cry2A protein had the best solubility in NEE lysate（p H 12.0）.The results of insecticidal activity analysis showed that Cry2Ab29 had the best insecticidal activity against newly hatched larvae of Spodoptera frugiperda,with median lethal concentration(LC₅₀)value of 42.5μg/g.2.The structure-activity relationship between Cry2A protein and insecticidal activity against Spodoptera frugiperda was preliminarily discussed.In order to verify the effect of the difference between the protein amino acids on insecticidal activity,the amino acid sequences of Cry2Ab29 with high insecticidal activity and Cry2Ah1 and Cry2Ad2 with low insecticidal activity were analyzed.Six amino acid difference sites were screened in domain III.The results of SWISS-MODEL analysis showed that 5amino acid sites were located on the surface.Based on the p HT315-cry2Ab29recombinant plasmid,site directed mutagenesis were performed on the selected different amino acid sites.6 soluble mutant proteins were successfully obtained,which were Cry2Ab29-H475A,Cry2Ab29-S482A,Cry2Ab29-N528A,Cry2Ab29-V568A,Cry2Ab29-T604A and Cry2Ab29-P616A.The results of insecticidal activity showed that the insecticidal activity of four mutant proteins against Spodoptera frugiperda was significantly improved.Among them,Cry2Ab29-V568A has the best insecticidal activity,with an LC₅₀ value of 8.47μg/g.Compared with Cry2Ab29,the insecticidal activity of Cry2Ab29-V568A increased by 6.87 fold.3.The mechanism of the difference in insecticidal activity between Cry2Ab29 and Cry2Ab29-V568A against Spodoptera frugiperda was discussed.To verify the relationship between changes in insecticidal activity and protein stability,Cry2Ab29and the mutant proteins were incubated with the midgut fluid of the third-instar Spodoptera frugiperda.The results indicated that the increase in insecticidal activity against Spodoptera frugiperda may not be related to the protein stability of the mutant.The saturated binding between Cry2Ab29 or mutant proteins and BBMV was further analyzed by Enzyme-linked immunoassay（ELISA）.The results show that Cry2Ab29and mutant proteins could bind to BBMV,and this binding could be saturated.The affinity constant（Kd）was calculated by Sigma-Plot:Kd _Cry2Ab29=489.93±59.64nmol/L,Kd _{Cry2Ab29-V568A}=19.74±8.42 nmol/L.Compared with wild-type Cry2Ab29,the binding ability of mutant V568A to BBMV is increased by about 24.8 times.The results of this study showed that a single point mutation in domain III could successfully increase the insecticidal activity of Cry2Ab protein,which might be related to the increased binding ability to midgut BBMV.These results indicate that the Cry2Ab domainⅢmight be the relevant area of the insecticidal activity against Spodoptera frugiperda,which laid a foundation for further modification of Cry2Ab protein to improve insecticidal activity.