Regulation And Expression Of Cry1Ac Gene In Bacillus Thuringiensis

Bacillus thuringiensis （Bt） is a Gram-positive, spore-forming soil bacterium that belongs to the B. cereus group. Bt can form a spore and one or more parasporal crystals encoded by cry or cyt gene when environment is poor. B. thuringiensis subsp. kurstaki HD73consists of cry1Ac gene, the crylAc gene is controlled by the regulatory factors SigmaE and SigmaK.The preliminary study of our group indicated that crylAc promoter had weak transcriptional activity in transition phase. The research of truncated cryl Ac promoter showed that317bp to368bp ATG upstream and0to252bp downstream were the key sequences to improve the transcription activity. The research gave an evidence that crylAc gene was complex in regulation and amounts of potential regulatory factors and mechanisms need a further exploration.The research found the proteins that could have interaction with crylAc promoter by DNA affinity purification. We found three transcriptional regulators HD73₃743, HD73₀689and HD73₀242may have interaction with crylAc promoter. The products of HD73₃743and HD73₀689were ArsR family transcriptional regulators possibly. The product of HD73J3242was redox-sensing transcriptional repressor Rex possibly. HD（Δ3743）, HD（Δ0689） and HD（Δ0242） mutants were obtained through the method of homologous recombination technology, respectively. The growth curve of three mutants in LB and SSM showed that three genes deletion did not effect the growth. The promoter of cry1Ac was fused with the lacZ gene, and then the recombination plasmid was transformed into HD73strain, HD（Δ3743）, HD（Δ0689） and HD（Δ0242） mutants, respectively. The β-galactosidase assay demonstrated that HD73₃743, HD73₀689and HD73₀242genes deletion did not effection the transcription activity of crylAc promoter. Sporulation efficiency assay demonstrated that HD73₀689and HD73₀242genes deletion did not effect the sporulation formation.The research attempt to explorat the crylAc gene direction by the non-cry gene promoter. The exsYgene encoding component protein of exosporium basal layer in Bacillus thuringiensis, that is homologous with cotZ in Bacillus subtilis, and the exsY promoter transcribes on the late sporulation phase,. Which is controlled by the regulatory factor σK. The research utilizes the exsY promoter to express crylAc gene in Bacillus thuringiensis. The crylAc gene was directed by the non-cry gene exsY promoter and was expressed in the acrystalliferous mutant HD73-. Transmission electron microscope （TEM） was used to observe the formation of crystal inclusion. This study provides a new idea for Cry crystal protein expression, and our finding provides a potential application for the genetically modified engineered Bt strains.