Abstract:[Objective] Compared to traditional Bt strains, Bacillus thuringiensis strain LM1212 can differentiate into spore-formers and crystal-producers. In this study, we tried to reveal the effect of plasmid curing on the cell differentiation of LM1212 strain. [Methods] The endogenous large plasmids of LM(p35'Z) carrying cry35-like gene promoter and lacZ gene fusion plasmid p35'Z were cured by high temperature treatment. The mutants were selected at HCO plates with X-gal and identified by using primers of cry genes. We extracted plasmids of mutants and analyzed them by pulsed field gel electrophoresis (PFGE). Observation by laser confocal scanning microscope and optical microscope, calculation of spore formation rate, SDS-PAGE and LC-MS/MS (Q-TOF) mass spectrometry were used to determine the effect of plasmid curing on the cell differentiation and expression of cry genes in LM1212 strain. [Results] Two plasmid curing mutants LM(p35'Z)-W and LM(p35'Z)-DB strains were obtained. The colony of LM(p35'Z)-W was white and LM(p35'Z)-DB was dark blue on HCO plates containing X-gal. It indicated that the activity of cry35-like gene promoter was affected in these two strains. Cell morphology observation showed that more crystal-producers were found in LM(p35'Z)-DB than in both LM(p35'Z)-W and LM(p35'Z). Crystal protein production in LM(p35'Z)-DB increased, but not in LM(p35'Z)-W. [Conclusion] Plasmid curing affected cell differentiation of LM1212 strain Cry protein expression. This will provide the excellent genetically materials for better revealing the regulation mechanism of cell differentiation in LM1212 and genetic modification of Bt strain.
