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

The bacterial strain B-02,isolated from the tomato infected by Botrytis cinerea in Zibo,was a Bacillus cereus strain effective to control Bo.cinerea.To research the antagonizing mechanism of Ba.cereus B-02 against Bo.cinerea at molecular gene level, the antibiosis-free mutant was constructed using transposon tagging technique,and then integration sites and flanking sequences were identified by TAIL-PCR(thermal asymmetric interlaced PCR).It was aimed at to find new antibacterial functional genes and then analyze the regulation mechanism of this phenotype.In this study,pTV1,a temperature-sensitive suicide vector with Tn917,was introduced by electroporation into Ba.cereus B-02 in the condition of 9.0-12.5kv/cm, 200Ω,25μF.17 transformants with chloramphenicol resistance were obtained and this property was highly stable and inheritable,which was proved by PCR and plasmid electrophoresis.By constant high temperature(44.5℃) mutation,the transposon Tn917 was successfully inserted into the genome of Ba.cereus B-02 and 1670 mutants were obtained resistant to erythromycin and lincomycin but susceptible to chloromycetin.In this study,the average efficiency of transposition was 4.29×10^-4,no replication fusant was appeared.By successive transfer culture,Tn917 was still inherited in the mutant strains proved by PCR and southern blotting.Among 1670 mutants,1 antibiosis-free mutant was obtained no more antagonizing Bo.cinerea,named as B-02-T.Southern blotting proved that transposon Tn917 mutagenesis resulted in random genome insertions in.Southern blotting analysis showed that Tn917 was randomly integrated into the genome of Ba. cereus B-02 strain,with single insertion copy.The flanking gene fragment of Tn917 insertion site was cloned by TAIL-PCR amplification using the genome of mutant strain B-02-T.After sequencing and gene splicing of the upper and downstream fragment by removing Tn917 vector sequence,a 2705 bp gene fragment was obtained.Homology alignment revealed that this sequence showed high homology to the plasmid gene of Bacillus which expressed hypothetical protein.From the above,it could be infered that the target gene was a new antibacterial functional gene.