Expression of cry1Ac gene directed by PexsYpromoter of the exsY gene encoding component protein of exosporium basal layer in Bacillus thuringiensis

Abstract:

Abstract:［Objective］To discover new elements for cry gene expression，PexsY，which is the promoter of the exosporium basal layer structural gene exsY，was used to express cry1Ac gene in Bacillus thuringiensis．［Methods］We used be tagalactosidase assays by promoter-lacZ fusion to analyze the transcriptional activity of exsY promoter and truncated exsY promoter．The cry1Ac gene was directed by the non-cry gene promoter PexsY and was then expressed in Bacillus thuringiensis HD73．Transmission electron microscope (TEM) was used to observe the formation of crystal inclusion．The Cry1Ac yieldswere evaluated by protein quantification and SDS-PAGE analysis．Bioassays against Ostrinia furnacalis were used for the functional verification．［Results］ Beta-galactosidase assays showed that the exsY promoter had a strong transcriptional activity in the acrystalliferous mutant strain HD73－on the late sporulation phase．Cry1Ac expression products directed by the PexsY could form diamond crystals．SDS-PAGE analysis showed that the cry1Ac gene directed by the cry8E promoter has the highest protein yield among the four promoters while the cry1Ac gene under the direction of PexsYorcry3A promoters showed similar protein yields．The bioassay results showed that the Cry1Ac protein directed by the PexsY promoter was toxic against Ostrinia furnacalis．［Conclusion］The cry1Ac gene under the direction ofthe non-cry gene promoter PexsY was able to express the Cry proteins at the late sporulation phase and could form crystal inclusion in a B．thuringiensis strain．Our finding provides applicationpotential for the genetically modification of engineered Bt strains．