Regulation And Expression Of Cry Genes In Bacillus Thuringiensis HD12

Bacillus thuringiensis(Bt) is a Gram-positive bacterium that widely spread in nature. It can be produce the parasporal crystal which consist of insecticidal proteins during sporulation.The insecticidal crystal protein is encoded by cry genes or cyt genes. The application of B. thuringiensis pesticides is associated with several challenges such as limited toxicity, narrow range of specificity of insecticidal spectrum, and the emergence of resistance. Therefore, the construction of a strain with synergistic expression of multiple insecticidal genes is necessary in order to expand the insecticidal spectrum of such pesticides and to increase their activity against target pests.B. thuringiensis subsp. morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, included eight cry genes, as indicated by genome sequencing. This strain produced crystals that are toxic to lepidopteran species.It was fit for the expression of multiple insecticidal genes.These crystal inclusions were purified by sucrose gradients and SDS-PAGE, followed by liquid chromatography-mass spectrometry, and found to contain five proteins(Cry1Da, Cry1 Ae, Cry1 Bb, Cry1 Fb, and Cry1Ja). The ?-galactosidase activity of the cry1 Da, cry1 Ae, cry1 Bb, cry1 Fb, and cry1 Ja promoters were analyzed in strain which different genetic background. It was indicated that transcription of cry1 Da is controlled by SigE; however, the other four cry genes were found to be controlled by both SigE and SigK. The activity of the cry1 Ja and cry1 Fb promoters was the strongest among the five genes studied. These promoters were therefore used to direct the expression of the Cry1 Ac and Cry2 Ab. By the methods of SDS-PAGE and liquid chromatography-mass spectrometry,the expression of the Cry1 Ac and Cry2 Ab was accurately guided by the promoters of cry1 Ja and cry1 Fb. The strain of multiple cry genes which could be expressed by two different promoters with the same transcriptional mechanisms has been constructed successfully.Our findings indicate that promoters with the same transcriptional profile may be used to direct the expression of different cry genes in one strain. Our results are expected to be valuable for the construction of strains with efficient expression of multiple cry genes in order to overcome current limitations associated with the application of B. thuringiensis-based insecticides. This study provides novel insights into the modulation of multiple cry genes, whose transcription is regulated by similar mechanisms, from a single B.thuringiensis strain.