The Cloning And Identification Of Virulence Gene Of Xanthomonas Campestris Pv.Campestris

Xanthomonas campestris pv. campestris(Xcc) is an important pathogen of cruciferous plants, it causes the black rot disease of cruciferous crops in the worldwide. One of product extracellular polysaccharide(EPS) named xanthan gum of Xcc is wide range used in industry. For the great economic value , a lot of research on the pathogenesis and the mechanism of EPS biosynthesis of the bacteria have been done. The sequencing of the complete genome sequence of Xcc 8004 strain have been accomplished and the transposon Tn5gusA5 inserted mutant library of the strain also have been constructed by our research team recently. In this study, the virulence of 17280 strain of Tn5gusA5 inserted mutants of 8004 strain are tested by clipping inoculation in leaves of radish, and 226 strain of virulence deficient mutants are isolated, the inserted gene of some of the virulence deficient mutants are cloned by TAIL-PCR, and 4 virulent genes have been identified by gene deletion and completement.The virulence of 5 inserted mutants of wbxL gene lost by clipping inoculation, and the deleted mutant of the gene is constructed, the virulence of the deleted mutant is lost also, and the growth of the deleted mutant in the host is also repressed, but the EPS yield and extracellular enzymes activity have no change, the virulence of the deleted mutant can be resumed when the mutant is completed in trans by a PCR fragment containing wbxL gene. Sequence analysis shows that wbxL gene similar to the wbpL gene of P. aeruginosa, and the deduced amino acid sequence of the gene also has the glycostransferase 4 family domain.The deleted mutant of waxE gene is constructed. The virulence of the mutant lost greatly, and the EPS yield decrease 35% than that of the wilt type strain 8004, the growth of the deleted mutant in the host is also repressed, but the extracellular enzymes activity have no change, the virulence and the EPS yield of the deleted mutant can be resumed when the mutant is completed in trans by a PCR fragment containing waxE gene. Squence analysis shows that waxE gene similar to the kdtX gene of S. marcescens and waaE of K. pneumoniae, the similarity is 52% and 50% respectively, and the deduced amino acid sequence of the waxE gene also has the glycostransferase 2 family domain.The deleted mutant of wxcA gene is constructed. The virulence of the mutant lost greatly, not only the LPS synthesis is affected but also the EPS yield is decread 50% or so, and the growth of the deleted mutant in the host is also repressed, the virulence and EPS, LPS synthesis can be resumed when the mutant is completed in trans by a PCR fragment containing wxcA gene.Squence analysis shows that wxch gene similar to the lpsE gene of C. crescentus and the similarity is 44%, the deduced amino acid sequence of the gene also has the glycostransferase 1 family domain.The virulence of 4 inserted mutants of ppsA gene lost, The deleted mutant of the gene is constructed and the virulence of the deleted mutant is lost also, the growth of the deleted mutant in the host is repressed, but the EPS yield and extracellular enzymes activity have no change, the virulence of the deleted mutant can be resumed when the mutant is completed in trans by a PCR fragment containing ppsA gene. Results show that ppsA gene is a virulence gene of Xcc.