The Role Of GerE Gene From Bacillus Thuringiensis

Bacillus thuringiensis(Bt) is most widely used as insecticidal microbe due to its efficient, harmless for human and other merits. The distinguished characteristics of Bt, comparing with the other Bacillus species, is its ability for producing insecticidal crystal proteins (ICPs), which are encoded by the cry or cyt genes. The gene expression and regulation in sporulation in Bacillus subtillis, as a model, were well studiedy. The expression and regulation of B. thuringiensis cry gene in early stage of sporulation were also studied as well. However, little is known about it in late stage of sporulation.There are four global transcriptional factorsσ~E, SpoIIID,σ~K and GerE, which are the critical temporal regulation factors in mother cell of sporulation, control expression of many genes during sporulation. As the sigma factors, theσ~E andσ~K combined with the core RNA polymerase(RNAP) recognize the special sequences of promoters and start transcription. The SpoIIID and GerE, the small binding proteins, regulate the transcription of genes which started by the RNAP withσ~E orσ~K. However the effect of GerE on the transcription and expression of cry gene is still not clear.In this thesis, the mutant strain named HD(ΔgerE) which knockout the gerE gene of HD-73 strain of Bacillus thuringiensis species has been constructed by the method of homologous recombination technology. the GerE overexpression fusion plasmid by inserting the gerE gene and its promoter sequence into the pHT315 which is a high-copy type plasmid in Bacillus thuringiensis was obtained. The GerE overexpression strain named HD(ΔgerE::gerE) was produced by transforming the GerE overexpression fusion plasmid into the HD(ΔgerE). Transcriptional analysis showed that the gerE gene was expressed in later stage of sporulation and the expression gerE gene maintained high level during the late stage of sporulation. On the side of phenotype, the cracking time appears delayed longly owing to lacking of gerE gene, likewise the amount of live buds cell and the spore germination rate reduce seriously rather than the original strain, and gerE gene overexpression can also result the amount of live buds cell decreasing to a low level. On the transcriptional activity of genes, in the case of deletion of gerE gene, the transcriptional activity of cry gene do not show significant modification, but the transcriptional activity of sigK gene go up to about two fold comparing to normal level. Interestingly, the gerE gene appears no transcriptional activity under the case of lacking of sigK gene. However, The expression of cry gene seems no difference between the original strain and the gerE mutant strain.