The Transcriptional Regulation Of Cry1Ac Gene In Bacillus Thuringiensis

Its ubiquitous ability to produce parasporal crystals with insecticidal activity distinguishes from the best-studied spore-forming bacterium Bacillus, which is the most important character to separate Bacillus thuringiensis from other Bacillus. B. thuringiensis have evolved two different cycles related to nutrient stress: vegetative cycle and sporulation. Accordingly, it is feasible to distinguish these cry genes into two different types: sporulation-dependent cry genes and sporulation-independent cry genes. Most known crystal protein genes are considered to be sporulation-dependent genes. The cry1Ac gene is considered as a typical example of the sporulation-dependent cry gene, controlled by the regulatory factorσE andσK. Cry1Ac-GFP fusion protein was constructed to study the relationship between the expression and regulation of cry1Ac and the spore formation.By gene fusion expression technology, the cry1Ac-gfp fusion expression plasmids with different copy numbers, pHT304-cry1Ac-gfp and pHT315-cry1Ac-gfp, were constructed. The pHT304-cry1Ac-gfp and pHT315-cry1Ac-gfp were transferred into the crystal-negative strain HD-73 cry- by electroporation, named HD-73-(304CG) and HD-73-(315CG). By homologous recombination technology, situ-fusion cry1Ac-gfp expression strain HD-73Φ(CG) was constructed. The results showed that the Cry1Ac-GFP fusion protein can be correctly detected by laser confocal microscopy in HD-73Φ(CG), HD-73-(304CG) and HD-73-(315CG). SDS-PAGE and Western analysis shows that Cry1Ac-GFP fusion protein can be detected in HD-73-(315CG) at t2. This result suggested that the expression of cry1Ac may start at the transition phase.To analyze the expression and regulation of cry1Ac, HD-73-(304CG) and HD-73-(315CG) is cultured in rich medium LB. The observation with laser confocal microscopy showed that the expression of Cry1Ac-GFP protein in HD-73-(315CG) can be detected at t6 during the transition phase. Western results showed that at t-1, t0 and t1 in HD-73-(304CG), HD-73-(315CG) and HD-73. The expression of Cry1Ac-GFP fusion protein can be detected. This means that cry1Ac has expressed during transition phase.Overlay-promoter (BtI and BtII) of cry1Ac is controlled by spore formation regulatorσE andσK. Spo0A is the key regulator for sporulation. HD-73(Δspo0A) mutant was contructed by homologous recombination. Fusion expression plasmid pHT315-cry1Ac-gfp was transformed into HD-73(ΔSpo0A) and HD-73(ΔsigE) by electroporation. The observation with laser confocal microscopy showed that the Cry1Ac-GFP fusion protein can be detected in HD-73(Δspo0A) and HD-73(ΔsigE). These results proved that Cry1Ac-GFP in log phase and transition phase was not controlled by the spore formation regulator Spo0A andσE.PlcR is a pleiotropic regulator which activates the transcription of many virulence factors outside the cell during transition phase in B. thuringiensis. HD-73(ΔplcR) mutant was constructed by homologous recombination. The PlcR did not regulate the Cry1Ac production. To study the recognize site of the cry1Ac regulators in log phase and transition phase, the truncation promoter (-1~134) of cry1Ac promoter plasmid pHT304-T1Acp was constructed. The results showed that the transcription activity of the truncation promoter was higher than that of 368bp promoter. This result proved that some inhibitor exist in upstream (from -134 to -368) of cry1Ac gene promoter.