Insertion Mutagenesis In Ralstonia Solanacearum Strain Isolated From Pogostemon Cablin Benth. By Tn5 Transposon

Bacterial wilt caused by Ralstonia solanacearum is the most destructive disease and causes severe losses in Pogostemon cablin Benth.The pathogen isolates were obtained from the diseased plant tissues in our previous study.In this study,identification of the isolates was conducted via PCR amplification,using specific primers based on 16S rRNA gene sequences of R.solanacearum strain GMI1000,and 12 strains were identified as R.solanacearum.And then,the genetic diversity among them was studied by repetitive-element PCR fingerprinting(rep-PCR).The strain PRS-84 was further used for Tn5 transposon mutagenesis by electroporation.The competent cells were prepared and electroporated with Tn5 transposon.The kanamycin-resistant clones were isolated and positively identified by PCR using primers based on kanamycin resistance gene.Tn5 transposon insertion-site flanking fragment of selected mutants were amplified by inverse PCR,and flanking sequences were obtained after sequencing.Preliminary bioinformatics analysis has been conducted on these sequences.1.Review on relevant literature in domestic and abroadRecent studies on identification,germplasm resources,cultivation techniques and main diseases control of Pogostemon cablin Benth.were reviewed.The diversity of host plants,races,biovars and phylotypes of Ralstonia solanacearum were also summarized.Besides,researches on structure,transposition mechanism of Tn5 transposon and its application in bacteriology were reviewed.2.Genetic diversity of Ralstonia solanacearum strains isolated from Pogostemon cablin Benth.A number of the pathogen isolates were obtained from the diseased plant tissues of Pogostemon cablin Benth in our previous study.In this paper,identification of the Ralstonia solanacearum strains were conducted via PCR amplification,using specific primers designed based on 16S rRNA gene sequences of R.solanacearum strain GMI1000,and 12 strains were identified as R.solanacearum.Further,differences in the colonial morphology of the R.solanacearum strains were observed on Triphenyl tetrazolium chloride(TTC)medium,and genetic diversity among them was analyzed by rep-PCR fingerprinting with three groups of primers which were BOX AIR;ERIC 1R,ERIC 2 and REP 1R,REP 2-1.The results indicated that R.solanacearum strains could be divided into six groups according to their colonial morphology,and the rep-PCR fingerprinting technique revealed that R.solanacearum strains can be divided into 8,7,7 groups based on BOX-PCR,ERIC-PCR and REP-PCR fingerprinting,respectively.The results also show that BOX-PCR is more efficient in identifying similarities and differences among R.solanacearum strains,and it confirmed that genetic diversity exists within R.solanacearum populations.3.Insertion mutagenesis by Tn5 transposon and the determination of flanking sequence in mutantsIn this study,the strain PRS-84 was used for Tn5 transposon mutagenesis by electroporation.The competent cells were prepared and electroporated with Tn5 transposon.The kanamycin-resistant(Kanr)clones were isolated and positively identified by PCR,using specific primers designed based on kanamycin resistance gene.PCR products of the positive colonies carried Kanr gene fragment with 663-bp.And then,genomic DNA was isolated from the mutants and digested with restriction enzyme Hind III,and the digested DNA was self-ligated with T4 DNA ligase.Futher,Tn5 transposon insertion-site flanking fragment of selected mutants were amplified by inverse PCR,and the flanking sequences were obtained after sequencing by the unlabeled reverse transposon-specific primers KAN-2 RP-1,and a total number of 538 flanking sequences of mutants were obtained.4.Bioinformatics analysis of the insertion sites in the mutantPreliminary bioinformatics analysis on the flanking sequences has been conducted.The 19-bp Tn5 transposon recognition sequence(ME)on the amplified PCR products confirmed that isolated mutants were a ’true’ transposon insertant.And then,the flanking sequences were compared to the nucleotide sequence database using the Blastn algorithm.Analysis of insertion sites and related gene function in PRS-84 genome were further carried out.The results showed that some mutants possessed the same insertion site and disrupted gene.Among the disrupted genes in the mutant,almost 400 of them were structural gene,approximate 60 of them were regulator and control gene,and approximate 40 of them were ribosomal RNA gene or transfer RNA gene.Several pathogenic related genes were disrupted by Tn5 transposon in the genome of PRS-84 strain,the gene expression products of the pathogenic related genes includ glucose-inhibited division protein A,GntR family transcriptional regulator,type II secretion system protein G and LysR family transcriptional regulator.