Cloning And Bioassay Of Genes Associated With The Virulence Of Bacillus Thuringiensis HBF-18Strain

White grubs are the larvae of Scarab, which is the largest group of underground pests, as well asthe most harmful one and resulting in the most loss. Bacillus thuringiensis (Bt) is one of the mostentomopathogenic microorganism studied and widely used. Bt strain HBF-18, isolated by Institute ofPlant Protection, Hebei Academy of Agricultural Sciences, had insecticidal activity against Holotrichiaoblita and Holotrichia parallela. cry8Ga1gene was cloned in our lab from this strain before. Cry8Ga1was toxic to Holotrichia oblita and Holotrichia parallela, but the toxicity of it was lower than that ofthe original strain HBF-18(only one50thand one70threspectively). The other cry8genes toxic to grubscloned in our lab didn’t show this trait. So, speculation that there are other insecticidal genes in HBF-18strain besides cry8Ga1was done. By traditional identification methods, we did not find any newinsecticidal genes in HBF-18strain. In current study, according to the results of the genomic sequencingthree novel insecticidal genes were cloned successfully from HBF-18strain, subsequently, insecticidalactivities of these new genes were conducted. The following were the main results.Based on genome sequencing of HBF-18strain, three new genes, cry8-like, vip1and vip2werefound from strain HBF-18through bioinformatics analysis, afterwards, cry8-like, vip1and vip2geneswere cloned respectively. cry8-like gene with2214bp contained a intact open reading frame, deducing738amino acid residues, and the similarity of Cry8-like is the closest with that of Cry8Db sharing51%.There were three domains necessary to insecticidal activity. On the upstream of cry8-like gene, therewere several binding sites of transcript factors Sigma A, Sigma D, Sigma E and ribosome binding site.On the downstream of that gene, there were two reverse repeat sequences. This gene was different fromother cry8gene with3.5kb full length.vip1gene, full length2634bp was deduced as878amino acid residues with molecular mass of98.1kDa, the similarity was76%, the closest with Vip1Ba. There was a sigal peptide in Vip1polypeptide with31amino acid residues from No.1-31. vip2gene, full length1386bp was deduced as462amino acid residues with molecular mass of52.6kDa, the similarity was88%, the closest withVip2Ac. There was a sigal peptide in Vip2polypeptide with30amino acid residues from No.1-30. vip2gene was located on the upstream of vip1gene, and there were7base pairs between them. On theupstream of vip2gene, there were binding sites of transcript factors Sigma B and Sigma G; on thedownstream of vip1gene, there was one reverse repeat sequence. The results of reverse transcriptionPCR analysis indicated that all the three genes could transcript in HBF-18strain normally.vip1, vip2, cry8-like genes were expressed successfully in E. coli Rosetta (DE3) strain, SDS-PAGEanalysis results showed that vip1gene was expressed as a protein with molecular mass of98kDa, vip2gene was expressed as a protein with molecular mass of52kDa, while cry8-like gene was expressedresulting in75kDa protein. In order to express the cry8-like gene in Bt strain,3′terminal of cry8Ea genewas fused to the end of cry8-like gene by the overlapping PCR, resulting in a cry8full length gene with3528bp, named cry8X gene. Then vip1, vip2, cry8X gene were transformed into Bt acrystalliferousmutant HD73-respectively, Three proteins were expressed successfully in HD73-with80kD of Vip,40 kD of vip2, and133kD of Cry8X, and engineered Bt strain named HDVIP1, HDVIP2and HD8X wereobtained, respectively. To evaluate the function of these new genes, bioassay for the insecticidal activityof HD8X, HD8G, HD8X+HD8G, HDVIP1+HDVIP2, HDVIP1+HDVIP2+HD8G and HBF-18strains or strain combinations to Holotrichia parallela larvae were performed respectively. The resultshowed that both HDVip1+HDVip2and HD8X had insecticidal activity against Holotrichia parallela,their LC50were3.51×1011å'Œ6.66×1011CFU/g soil respectively, and, their insecticidal activity werelower than those of HD8G and HBF-18strains. The synergistic factor of HDVIP1/HDVIP2/HD8G was4.15, revealed synergism against Holotrichia parallela, the synergistic factor of HD8X/HD8G was1.86,there was additive effect between them. The significant difference analysis showed that there was nosignificant difference between HDVIP1+HDVIP2+HD8G and HBF-18strain. So, we could explainwhy the insecticidal activity of HBF-18was higher than HD8G, it was because there was vip1and vip2genes express in HBF-18strain besides cry8Ga gene, these genes could improve the toxicity of Cry8Ga.In addition, Cry8X protein had insecticidal activity against Anomala corpulenta larvae, but no activityagainst Plutella xylostella.In summary, three new insecticidal genes (vip1, vip2and cry8X) were cloned and characterized inthe current study. And the reason of the significant differences of toxicity between the strain HD8G(only express cry8Ga gene) and strain HBF-18was revealed. Futhermore, the study provides new generesources for the biological control of grubs.