Excavation And Functional Verification Of New Insecticidal Genes From Bacillus Thuringiensis In Guizhou Area

Bacillus thuringiensis(Bt)has been widely used in agricultural production,forest protection,garden control,and prevention and control of sanitary pests since it was discovered.At present,more and more Bt preparations and Bt crops have been widely developed and applied,and good control effects have been achieved in the control of pests.Since the 1920 s,Bt protein has been used for the prevention and control of agricultural pests and has achieved excellent results.Therefore,isolation and screening of high-efficiency,broad-spectrum insecticidal activity of Bt strains,cloning of new insecticidal activity genes,provide new resources for the production of disease prevention and pest control.In this study,a series of Bt strains were isolated and screened by extensively collecting soil humus in Guizhou,and genotypes and insecticidal activities were analyzed,a batch of Bt strains with insecticidal activity against lepidopteran pests were obtained,three new Bt genes were cloned from two strains of CS137-2 and CS72-1.A total of 1170 samples were collected from various regions of Guizhou for the isolation of Bacillus antibiotics.A total of 976 Bacillus species were identified and 181 Bt strains were identified,the average isolation rate exceeded 18%.Genotypic analysis was performed on the more than 100 Bt strains obtained,a total of 15 genotypes including cry1,cry2,cry4,cry8,cry9,cry20,cry22,cry28,cry30,cry31,cry37,cry40,cry46,cry52 and cyt2 were found,87 unidentified genotypes.There are 31 different combinations of genotypes among the strains,and the most genotype-combined strains kill the lepidopteran pests,in addition,a combination of genotypes of Homoptera/Lepidoptera and Coleopteran/Diptera was also found.The expressed proteins of most Bt strains showed multiple bands under SDS-PAGE analysis.The standard protein Marker was used as the reference for the expression of 30-130 KDa protein;microscopic observations also revealed multiple crystal types,and most of the same strains contained two crystal forms.The shape of the parasporal crystals produced by the Bt bacteria is spherical,large and small diamonds,squares,and rice grains.Bioassay results showed that 101 Bt strains were active against Helicoverpa armigera and 80 strains had no insecticidal activity against Helicoverpa armigera.It shows that Bt resources in this area are abundant,the gene types are rich and varied,the insecticidal spectrum is wide,and it has good diversity.This study selected a wide variety of genes from the strains,the high insecticidal strains CS72-2 and CS137-2 were further studied.Through the whole genome sequencing and assembly of CS72-2 and CS137-2 strains,the PCR-RLFP cloning and PCR cloning of the two strains were designed based on the sequencing assembly results,and three new genes were cloned,it contains a second-level gene,a third-level gene,and a fourth-level gene.Named cry28-like,cry37-like,cyt2-like.After the gene cry28-like was constructed into the shuttle expression vector pSTK and transferred into the amorphous mutant HD73-,It has insecticidal activity against Ostrinia fumacalis and Helicoverpa armigera but for Caenorhabditis elegans and no insecticidal effect of Meloidogyne incognita.The functional verification of two insecticidal genes,cry37-like and cyt2-like,showed that the two induced proteins have insecticidal activity against two lepidopteran insects.The cry37-like and cyt2-like inducible proteins are active against.cry37-like inducible proteins have relatively low insecticidal activity against Caenorhabditis elegans and Meloidogyne incognita no insecticidal activity.The cyt2-like inducible proteins showed weaker insecticidal effects against Caenorhabditis elegans and Meloidogyne incognita,and the corrected mortality after 72 hours was 35% and 30%,respectively.