The Study On SigK Mutation In The Industrial Strain Bt-59 Of Bacillus Thuringiensis Var.israelensis

Bacillus thuringiensis var.israelensis?Bti?is an important biological factor used to control vector organisms such as mosquitoes and blackflies.However,there are some disadvantages of Bti in the practice of control of vectors,such as low virulence and short duration.In Bacillus,it had been proved that Sigma K??K?factor has the regulating functions of sporulation and mother cell lysis.Also,the formation of insecticidal proteins is affected by cascade regulation through Sigma???factors.In this paper,we studied the effect of sigK to Bti by constructed a Bti mutant strain of inactivating ?K factor in the industrial strain Bt-59,for the purpose of constructing of engineering bacteria strains of high virulence activity and lasting duration effect.The main findings are as follows:1.Construction of the sigK deletion mutant strain Bt59?AsigK?by homologous recombinationBy sequencing the whole genome of Bt-59 strain,it was determined that the sigK gene is located at Genome1₀5261,which is 714 bp in size and encodes 237 amino acids.The amino acid sequence Bt-59 sigK is 100%similar to those in the other reported Bt subspecies.Amplifyed the homology arms of sigK gene and ligated to pMAD,the knock-out vector pMAD?sigK was constructed.By transforming the vector into the Bt-59 competent strain,a sigK deletion mutant Bt59??sigK?was constructed by homologous recombination.A 1495 bp fragment PsigK-sigK containing the sigK gene and its promoter region was also cloned.This PsigK-sigK was introduced into the expression vector pHT315 and transformed into the mutant strain Bt59??sigK?,resulted in a genetically complementary strain Bt59?HFsigK?.2.Clarified the effects of the sigK mutation to the spore formation of Bt-59The growth of vegetative cells of Bt59??sigK?was insignificantly different from the original strain Bt-59.But there was no spore formation observed in the late stage of(Bt59??sigK?.To the sigK complementary strain Bt59?HFsigK?,the spore formation was restored,with the spores about 3.33×106 CFU/mL,which was equivalent to the original strain of 2.43×106 CFU/mL spores.It was indicated that the sigK deletion in Bt-59 caused the incapacition of forming spores.The sigK deletion also affected the mother cell lysis of Bti.The time of complete mother cell lysis of Bt59??sigK?was extended from T25 to T60,while Bt59?HFsigK?restored the mother cell lysis rate to the original level of Bt-59.3.Analysis the effect of sigK mutation to the expression and activity of insecticidal proteins of Bt-59Bt59??sigK?generated insecticidal crystal proteins in the late stage normally.The crystal morphology and size were not difference from that of Bt-59.SDS-PAGE showed that there were about 1.67 times of Cry4Aa/4Ba proteins and 1.21 times Cyt1Aa protein up-expression compared with Bt-59,while the expression of Cry11Aa protein had no change.Bioassay results showed that the sigK deletion mutation significantly reduced the insecticidal activity of Bt-59 against Culex pipiens pallens.But there was no obviously affect to the activity against Aedes albopictus.4.Explored the effect of sigK mutation to the hydrolase of Bt-59 CwlC:The hydrolyzed cell wall activity of Bt-59 CwlC was clarified by cloned a 732 bp fragment cwlC from Bt-59 strain and expression in BL.A 617 bp cwlC promoter region?PBcwlC?was amplifyed and ligated to pHT304-18Z to construct the recombinant vector pHTPBcwlC.By transformed the expression vector into the strain Bt59?AsigK?,the BcwlC-lacZ expression strain Bt59??sigK??PBcwlC-lacZ?was obtained.It was found no transcriptional activity of lacZ activity in Bt59??sigK??PBcwlC-lacZ?,indicated that the Bt-59 cwlC gene was controled by ?k factor.Compared CwlC of Bt-59 to that of Bt subsp.kurstaki HD73,there might be hydrolyzed functions,resulted in that Bt-59 CwlC could restore the HD?AcwlC?phenotype of cell lysis.