Cloning And Sequencing Of The Antagonism-related Gene Fragments Of A Broad-spectrum Antagonistic Enterobacter Cloacae B8

Using Enterobacter cloacae B8, the mutated strains B8B and B8F, and the recombinant clones pB and pF, we try to sequence the antagonistic-related genes of Enterobacter cloacae B8 by subcloning and Genome Primering System.The acquired sequences were analyzed with BLAST Program to find any homology to sequences deposited in Genebank. The results showed that the F fragment ,728bp in length, could be a new gene with a little homology to the genes coding for polyketide synthetase or fatty-acid synthetase and the B fragment, about 4kb in length, is inferred to have repeat sequences around Tn5 insertion site,in which there is homology to the WA 314 right arm of the high-pathogeniciry island of Yersinia enterocolitica.To reveal any pathogenicity of Enterobacter cloacae B8 and its mutated strains B8B and B8F to animals, the experiment with mice was carried out. The result showed that B8 and its mutated strain B8F had weak transient virulence but the mutated strain B8F had no virulence.The chromosome walking adapter consisting of 40nt and 44nt oligonucleotides was designed and synthesized. And the two pah's of PCR primers that bind to the adapter and the sequence of F fragment close by Tn5 respectively were also designed. The genomic DNA of B8 was isolated, digested with BamH I, and ligated to the adapter.Using the two pairs of the primers ,two rounds of PCR were performed hi turn and a fragment of 239bp was amplified successfully.lt was proved by cloning and sequencing that 18bp of the fragment is the sequence opposite to F fragment on the left of Tn5 insertion site in B8F,the other is part of the 728 bp of F fragment. This result makes it possible to continue to carry out chromosome walking ,to clone and sequence the wholegenes of B fragment and F fragment ,and to reveal the antagonistic molecular mechanism of B8.