| Bacillus subtilis is a beneficial soil bacterium for hunman to resist environmental, as it can produce stress and heat tolerance spores. It can also antagonize a variety of pathogens. So, it is commonly used for production, processing and storage of their biocontrol agents which has good application prospects. AbrB, which is one of the major regulatory proteins of Bacillus subtilis during spores generation process, can inhibit spores formation. Gene named AbrB were amplified by the polymerase chain reaction (PCR), using genomic DNA of Bacillus subtilis B579 which was screened in the previous research in this paper. And the sequence was analyzed further using genetic engineering methods to construct recombinant expression plasmids to achieve AbrB express in the Bacillus subtilis B579. And effects of AbrB induced on spores generate were studied, laying the foundation for the production of a high concentration of spore of biocontrol agents.The plasmid electroporation conditions of Bacillus subtilis B579 were optimized. It was shown that adding Glycine to a final concentration of 0.65 mmol/mL in the medium, collecting cell at OD600 0.8, concentrating the cells to a final density of larger than the original cell concentration 30, selecteing pulser voltage of 2 kV with lmm electroporation chamber, the electroporation efficiency reached 4×102 transformants/μg DNA.AbrB were amplified by the polymerase chain reaction (PCR), using Bacillus subtilis B579 genomic DNA as a template and the primers designed based on the reported gene sequences of AbrB of Bacillus subtilis 168. In addition, homology analysis indicated the cloned AbrB genes and the amino acid sequence encoded by it shared 93% and 100% identity with the reported target gene sequences of Bacillus subtilis 168 in NCBI databases, respectively. This protein belongs to the transcription initiation regulatory protein, which belongs to Antitoxin-MazE superfamily.The constitutive expression plasmid pHY-P43-AbrB expressing the regulatory protein was constructed based on plasmids of pHY-P43. and it was transformed into the B. subtilis B579.The regulatory protein AbrB was expressed successfully in the B.subtilis B579 using this plasmid. The protein molecular weight of AbrB approximately 10.77 kDa.The shake flask experiments showed that, compared with the original strain, spore generation start time of the recombinant strain B. subtilis B579 (pHY-P43-AbrB) extended three hours, spore generation rate decreased 73.08%. The result demonstrated that overexpression of AbrB inhibits B. subtilis B579 Spore formation.In addition, the Lactose-inducible expression plasmplasmid pDG148-AbrB expressing the regulatory protein was constructed based on plasmids of pDG148 and it achieved the induced expression of AbrB in the B.subtilis B579. Compared with the original strain,when the initial lactose concentration of the culture medium was 0.5 g/L, the bacillus generation start time of recombinant strain B. subtilis B579 (pDG148-AbrB) extended four hours, the concentration of bacteria increased 98.95% during the fermentation process. At the end of fermentation, the spore concentration reached 4.34 X 108 CFU/mL and the spore efficiency 75%, respectively, compared with the original strain improved 34.33%,9.31%. |
