Cloning And Expression Of Antagonism-related Genes Of Bacillus Cereus B-02

The bacterial strain B-02 was isolated from the greenhouse soil in which the tomato was infected by Botrytis cinerea in Zibo urban area in Shandong Province.It is a Bacillus cereus strain effective to control Botrytis cinerea.To research the antagonizing mechanism of Bacillus cereus B-02 to Botrytis cinerea at molecular level,three interbedded specific primers were designed according to the gene fragment(573 bp) amplified from genome DNA of Bacillus cereus B-02 Mutant,and they were combined with a primer bank consisting of five short random degenerate primers to respectively amplify 5' and 3' flanking sequences from genomic DNA of Bacillus cereus B-02 by TAIL-PCR.A 2 344 bp (MZ-BcP) fragment was obtained by extracting,sequencing,shearing and splicing PCR products.The homology search found that MZ-BcP nucleotide sequence was more closely homologous with plasmid of Bacillus,which encoding putative replication protein.Five ORFs including the Tn917 insertion site were obtained and translated by DNAStar Analysis.One 585 bp ORF encode a 22.3 kD protein.It might be a novel gene or a regulatory factor which related antibacterial activity.In order to verify the interactions between 585 bp ORF gene fragment and antagonistic activity of Bacillus cereus B-02,585 bp ORF gene fragment was amplified by PCR,and subcloned into prokaryotic expression vector pET-32a(+),then the recombinant expression plasmids(pET-32a(+)-MZF585) were successfully constructed.It was transformed into E.coli BL21(DE3),A fusion protein of 42.3 kD was expressed after induced by IPTG induction,which was identical to the theoretical value.