Objective: To construct and characterize a sigK gene disruption mutant of Bacillus thuringiensis and to study influence of sigK gene disruption on the activation of cry3A gene promoter.
Methods: We constructed the sigK gene disruption mutant HD delta sigK by inserting kanamycin resistance gene via homologous recombination. Scanning electron microscopy and spore formation analysis were used to detect the abilities of sporulation and crystal protein formation of both the mutant and the wild-type strain. SDS-PAGE analysis was used to detect the expression of crystal protein. Beta-galactosidase assay of cry3A'-lacZ gene fusion was performed to analyze the influence of sigK gene disruption on the activation of cry3A promoter.
Results: The growth curve showed that mutant grew slowly in late stationary phase compared to the wild-type strain. Scanning electron microscopy and spore formation analysis indicated that no spore was produced in sigK disruption mutant. SDS-PAGE results exhibited that the expression of cry gene was significantly decreased in the mutant. Beta-galactosidase assay showed that the activation of cry3A promoter was stronger in the mutant than that in HD-73 during late stationary phase, but the disruption of sigK gene had no significant influence on the production of Cry1Ac which was initiated by cry3A gene promoter.
Conclusion: These results indicated that sigK gene was one of the essential genes during the sporulation of Bacillus thuringiensis, and influenced the expression of crystal protein. The expression of crystal protein which was initiated by cry3A gene promoter in sigK disruption mutant could be used to develop high-efficiency and safe biological pesticides.