Activity of cry gene promoter of Bacillus thuringiensis LM1212

Abstract:

[Background] Bacillus thuringiensis (Bt) LM1212 with a unique differentiation phenotype has been identified. It carries 14 insecticidal genes, which comprise 10 transcription units. With the crystal producing cell regulator (CpcR) derived from the LM1212 strain and the cry35-like gene promoter activated by CpcR, the non-sporulating expression system of insecticidal protein has been successfully established in the typical Bt strain HD73. [Objective] To compare the transcriptional activities of different insecticidal gene promoters of strain LM1212, identify the promoters with high transcriptional activity and activated by the transcription factor CpcR, and optimize the non-sporulating expression system. [Methods] Ten promoter regions were respectively fused with lacZ reporter gene on the pHT304-18Z vector, and 10 recombinant plasmids were obtained. The cpcR gene and its promoter (P_cpcR-cpcR) were reversely integrated in the upstream region of each selected promoter region and the lacZ reporter gene, and 10 recombinant plasmids capable of expressing CpcR and corresponding to the above construction were obtained. Subsequently, these recombinant plasmids were separately transferred into the HD73 strain without CpcR, and thus 20 recombinant strains were obtained for the determination of β-galactosidase activity. The expression of insecticidal protein was determined by microscope observation and SDS-PAGE. [Results] In HD73 strain, the transcription of the promoters P₁, P₃, P₄, P₅, P₆, P₇, and P₈ could be activated by CpcR, while that of the promoter P₁₀ was inhibited by CpcR. In the presence of CpcR, P₇ and P₈ showed high transcriptional activities. Vip3Aa11 protein with strong activity against Spodoptera frugiperda was successfully expressed by CpcR combined with P₇ and P₈. [Conclusion] The screened promoter with high transcriptional activity can be used to optimize the non-sporulating expression system of insecticidal protein, so as to construct novel bacteria for the biocontrol of S. frugiperda.