| Despite the successful application of toxins from Bacillus thuringiensis as biological control agents against pests,pests are showing resistance against an increasing number of Bacillus thuringiensis toxins due to evolution;thus,new toxins with higher toxicity and broad-spectrum activity against insects are being increasingly identified.To find new Bt strains and insecticidal toxin protein genes,in this study,the new Bacillus thuringiensis strain Bt Gxmzu777-1,which was isolated from the soil samples of Wuzhishan scenic spot in Hainan province in the early stage,was subjected to biological characteristics analysis,whole genome sequencing analysis,and identified a predicted insecticidal toxin protein gene cry1Na2-like.Subsequently,the predicted insecticidal toxin protein gene cry1Na2-like was subjected to molecular cloning,expression and purification,and activity and function exploration,and the results were as follows:Bt Gxmzu777-1 was a Bacillus thuringiensis strain with good insecticidal activity against Pyrausta nubilalis(LC50273.63μg/mg)and was able to produce a large number of irregularly shaped satellite crystals upon spore release.SDS-PAGE analysis showed that about 70 k Da and27 k Da protein bands were expressed after trypsin digestion.Through sequencing analysis,sequence analysis,domain analysis,and screening prediction of the whole genome data,two toxic protein genes p1G＿81 and p4G＿288 were obtained.Among them,p1G＿81 contains complete Endotoxin＿N,Endotoxin＿M and Endotoxin＿C 3D toxin domains,and its homology similarity with Cry1Na2 is only 24.1%.It is preliminarily considered to be a novel toxin protein,and temporarily named Cry1Na2-like.Molecular cloning,vector construction,induced expression and purification of Cry1Na2-like were performed to obtain a target band protein with the size of about 50 k Da,and an active Pyrausta nubilalis with the size of about 35 k Da was obtained after trypsin digestion.Analysis of its insecticidal activity showed that both the purified protein and the active Pyrausta nubilalis had a toxic effect on Ostrinia furnacalis larvae,with an LC50of 3.626 mg/mg and 2.01 mg/mg,respectively.The middle intestinal tract of corn borer larvae infected with Cry1Na2-like at different time points was extracted for high-throughput sequencing and the abundance of intestinal flora was detected.The results showed that significant changes were found in dominant phylum at the phylum level,and the main genera at the genus level were Enterobacter and Enterococcus,and the abundance of Bacteroides spp.was significantly increased.These results will provide experimental reference for further study on the insecticidal activity,toxicity spectrum and synergistic effect of Bt Gxmzu777-1 strain and its internally predicted insecticidal toxin protein gene Cry1Na2-like. |
