Analysis On The Key Sites Of Insecticidal Activity Of Bacillus Thuringiensis Sip1Aa Protein

Secreted insecticidal protein?Sip?are a kind of insecticidal proteins secreted during the growth and metabolism of Bacillus thuringiensis,which have no homology with Cry proteins and Vip proteins,and have good insecticidal activities against Coleoptera pests.At present,little is known about the insecticidal mechanism of Sip protein.In this study,mutants were constructed by bioinformatics analysis to obtain the corresponding relationship between mutated amino acid sites and insecticidal activities.It has important research and application prospect to study the insecticidal mechanism,improve the insecticidal virulence of Bt and delay the resistance of insect.Bioinformatics research methods such as homologous modeling were used to preliminarily predict the three-dimensional structure of Sip1Aa protein,and some acidic and alkaline amino acid residues in its conserved structural domain were selected for study.PCR mediated site directed mutagenesis was used,with the methylated p ET-sip1Aa recombinant plasmid as the template,18amino acid sites?H99,K109,K128,E130,D134,D136,D141,K145,K193,H242,H259,D290,R292,D299,H303,H318 and D328?were mutated by alanine scanning,and 18 mutants were successfully constructed.SDS-PAGE analysis showed that all the 18 mutants were able to produce soluble protein of about 37.6 k Da in size,indicating that the mutations did not destroy the higher structure of protein.The insecticidal activity of mutated protein was qualitatively and quantitatively analyzed against Colaphellus bowringi Baly.The insecticidal activities of K109A,K128A and E130A against Colaphellus bowringi Baly were significantly improved,among which the insecticidal activities of K128A were the most significant.The LC₅₀ value of K128A was 0.18?g/m L,which was about 10 times lower than that of Sip1Aa protein.The LC₅₀ value of E130A was 0.286?g/m L,which was about 6 times lower than that of Sip1Aa protein.Moreover,both K128 and E130were in the?9-?10 Loop,which was speculated to be an important Loop in Sip1Aa protein.The mutants D290A,H242A and H303A were significantly less toxic to the Colaphellus bowringi Baly,and these three sites may be at the key sites of the insecticidal activity of Sip1Aa protein.The results of this study provide a theoretical basis for revealing the relationship between structure and function of Sip1Aa protein.In order to solve the problem that a large number of inclusion bodies are produced when Sip1Aa protein is expressed in E.coli,and to improve the soluble protein yield,this study constructed a expression vector of Sip1Aa protein,which was based on the p UC19 vector,guided by cry1Ac promoter and the overlapping PCR technique was used.To investigate the expression,insecticidal activity and solubility of Sip1Aa protein guided by the Bt cry1Ac promoter,and to compare with the Sip1Aa protein guided by the T7 promoter,and at the same time to preliminarily explore its fermentation conditions.The recombinant protein was successfully purified by adding a histidine tag to the recombinant plasmid by reverse PCR.The results showed that both cry1Ac and T7promoters could instruct Sip1Aa to express the soluble protein of 37.6 k Da,and there was no significant difference in the insecticidal activity of both.The soluble fraction of Sip1Aa protein guided by cry1Ac promoter was significantly higher than that of Sip1Aa protein guided by T7promoter.When the strain reached saturation,the fermentation time had little effect on the protein expression.It provides new ideas for the rapid expression of sip gene and insecticidal mechanism.In order to delay insect resistance,a random library of Sip1Aa protein was constructed by error-prone PCR.100 positive inverters were randomly selected for sequence determination,and a total of 25 mutants were generated,named M1-M25 respectively.A total of 29 base mutations occurred,with an average of 1.2 base mutations per mutant.Compared with the wild type Sip1Aa protein,the insecticidal activity of mutant M1?A31,Y118,D227?,M5?K168?and M21?I307?significantly decreased with the LC₅₀ value increased by 4-6 times.The mutant M8?R174S?with increased insecticidal activity against Colaphellus bowringi Baly was obtained,and its LC₅₀ value was reduced by 4 times.The results of this study provide reference for the molecular modification of Sip1Aa protein and the study of key sites of its insecticidal activity.