摘要
Ralstonia solanacearum strainYC45 isolated from Rhizoma Kaempferiae.The genome of YC45 was completely sequenced(Genbank NO.CP011997,NO.CP011998).A new gene ACH51_14495which has no homologous sequence with other organisms was found.ACH51_14495 was predicted to be a candidate of T3 SS effectors by T3 SS effector prediction website.In order to investigate the function of the gene ACH51_14495 in strain YC45,ACH51_14495 mutant of YC45 was constructed.Accordingto the upstream and downstream sequence of YC45 gene ACH51_14495,the PCR primers were designed to amplify the 500bp-fragments of upstream and downstream of ACH51_14495 gene,respectively.The fragments and gentamycin gene were cloned into suicide vector pK18 mobsacB,resulting in recombinant plasmid pK18-ACH51_14495-Gm.The plasmid was then introduced into R.solanecarum strain YC45.The ACH51_14495mutant,named YC45-△14495,was generated by homologous recombination and selected by three-steps method.The mutant was identified by PCR.After incubation at 30℃ for 2 days on tetrazolium chloride(TZC) medium,the mutant showed small,crimson colonies,while its wild-type strain exhibited large,irregular round,fluidal and white colonies with pink center.The results of hypersensitive reaction test showed that the mutant could not cause necrosis,but the wild-type strain YC45 might produce necrosis on five-leaf stage tobacco after 24 h post inoculation.Thus,the gene ACH51_14495 deficiency obviously affected bacterial colony morphology and hypersensitive reaction of YC45,and the gene was related with the pathogenicity of YC45.
Ralstonia solanacearum strainYC45 isolated from Rhizoma Kaempferiae.The genome of YC45 was completely sequenced(Genbank NO.CP011997,NO.CP011998).A new gene ACH51_14495which has no homologous sequence with other organisms was found.ACH51_14495 was predicted to be a candidate of T3 SS effectors by T3 SS effector prediction website.In order to investigate the function of the gene ACH51_14495 in strain YC45,ACH51_14495 mutant of YC45 was constructed.Accordingto the upstream and downstream sequence of YC45 gene ACH51_14495,the PCR primers were designed to amplify the 500bp-fragments of upstream and downstream of ACH51_14495 gene,respectively.The fragments and gentamycin gene were cloned into suicide vector pK18 mobsacB,resulting in recombinant plasmid pK18-ACH51_14495-Gm.The plasmid was then introduced into R.solanecarum strain YC45.The ACH51_14495mutant,named YC45-△14495,was generated by homologous recombination and selected by three-steps method.The mutant was identified by PCR.After incubation at 30℃ for 2 days on tetrazolium chloride(TZC) medium,the mutant showed small,crimson colonies,while its wild-type strain exhibited large,irregular round,fluidal and white colonies with pink center.The results of hypersensitive reaction test showed that the mutant could not cause necrosis,but the wild-type strain YC45 might produce necrosis on five-leaf stage tobacco after 24 h post inoculation.Thus,the gene ACH51_14495 deficiency obviously affected bacterial colony morphology and hypersensitive reaction of YC45,and the gene was related with the pathogenicity of YC45.
引文
