Function Analysis Of SpoⅢD Gene From B. Thuringiensis

Bacillus thuringiensis(Bt) is most widely used as insecticidal micriobe due to its high toxin,safety and the other merits.Comparing with the other Bacillus species,Bt can procude insecticidal crystal proteins(ICPs).The study on mechnism of crystal formation is mainly focused on the expression and regulation of cry gene encoding insecticidal crystal proteins.But the research about the relations between production of ICPs and sporulation genes was little.Previous study showed that cry1-type genes were transcribed by two overlapping promoters(BtⅠand BtⅡ) controlled by RNA polymerase containing sporulationσfactorsσ~E andσ~K in B.thuringiensis. Also,during sporulation of Bacillus subtilis,four regulatory proteins act in the orderσ~E,SpoⅢD,σ~K, and GerE to temporally control gene expression in the mother cell.σ~E andσ~K are transcription regulatedσfactors,SpoⅢD and GerE is a small,sequence-specific DNA-binding protein which activates or represses transcriptions of many genes.But the fuction of SpoⅢD and GerE protein as regulated factors during the expression cry1Ac genes is not clear now.This study focused on the effect of spores formation and expression of cry gene by deletion of SpoⅢD gene in B.thuringiensis.The SpoⅢD gene inactivation mutant was obtained by homologous recombination and its genetically complementary strain was also constructed.Scanning electron microscopy and spore formation analysis showed that nearly no spore was produced in the strain with SpoⅢD deletion while the crystal existed.SDS-PAGE results showed that the expression of cry gene was decreased in the mutant in LB medium and PB medium but not affected obviously in SSM medium. Beta-galatosidase assay indicated that transcriptional activity of pro-σ~K was decreased and that of pro-σ~E was increased in the mutant strain by fusions between two promoters and lacZ gene.Converse binding sequence analysis of SpoⅢD protein showed that no possible SpoⅢD binding sequence in cry1Ac promoter and gene.Therefore,we think that the change of insecticidal crystal protein expression in SpoⅢD gene deleted strains of should be attributed to the sum influence of pro-σ~E and pro-σ~K gene expression.preliminary analysis was also done about gerE gene,and the gene deleted vector was constructed. Conserve sequence analysis showed that sequence between -89 and -77 site before ATG of cry1Ac gene has the conserve binding sequence of GerE protein.More research is needed to explore the function of GerE on the transcription and expression of cry1 Ac gene.This study is important in the exploring of the relationship between transcriptional regulated factors and the expression of cry gene.Also,this new type of strain with SpoⅢD gene deleted which produces crystal protein while do not produce spores offers us a new thinking way of the construction project on higher eco-secure recombinant Bt strains.Our result also showed that through importing exogenous multi-copes of cry1Ac gene,the expression of insecticidal crystal protein was increase, which makes this strain better applied.Along this line of thought,we can continue to explore other genes related to spore formation,construct new recombinant strains with higher eco-security.