Genetic Stabi Lity Of Tn5 Transposon Insertion Mutants Of Ralstonia Solanacearum Isolated From Pogostemon Cablin

The Ralstonia solanacearum strains isolated from diseased plant tissues of Pogostemon cablin benth.were genetic mutagenized with Tn5 transposon by electroporation in our previous study.In this study,sixteen mutants from wild-type strain PRS-84 mutagenized with Tn5 transposon were cultured and passaged on NA medium and select medium containing Kanamycin for 100 times.Every five continuous passages,kanamycin-resistant(Kan,)gene in genomic DNA of mutants was identified by PCR using primers based on Kan' gene.Mutants subcultured for 100 passages were futher identified by southern blot hybridization.Tn5 transposon insertion-site flanking fragment of mutants subcultured for 100 passages was ampli fied by PCR using reverse transposon-specific primers,and DNA sequence of the flanking fragements were analyzed with chain termination method.Besides,colonial morphology of mutants during subcul ture was observed on Triphenyltetrazol ium chloride(TTC)medium,respectively.And the biological characteristics of mutant strains(PRS-84-6-13 and PRS-8 1-10-69)subcul t ured for 100 passages were invest igated.1.Review on relevant literature in domestic and abroadThe study on identification,germ]asm resources,cultivation techniques and diseased control in Pogostemon cablin benth.was reviewed.Researches on identification and pathogenic mechanism of Ralst.onia solanacearum and the control of bacterial wilt disease were summarized.Besides,researches on structure and transposable mechanism of transposon,identification methods of transposon insertion sites,the genetic stability of transposon insertion mutant strains and application of transposon mutagenes:i s were reviewed as well.2.Molecular identification of mutants subcultured for 100 passegesThe mutants from wild-type strain PRS-84 mutagenized with Tn5 transposon were cultured and passaged for 100 times on NA medium.Every five continuous passages,kanamycin-resistant(Kanr)gene in genomic DNA of mutants was identified by PCR using primers based on Kanr gene.The PCR result showed that Kanr gene existed in genomic DNA of mutants at different passages.Further,the number of copies and insert position of Tn5 transposon in mutants subcultured for 100 passages were identified by southern blot hybridization.The results showed that the Tn5 transposon was single copy insertion in these mutants and the insert fragement existed in genomes of mutants subcultured for 100 passeges.3.Sequencing of Tn5 transposon insertior-site in mutants subcultured for 100 passagesTn5 transposon insertion-site flanking fragment of primary mutants and mutants subcultured for 100 passages was amplified by reverse transposon-specific primers(KAN-2 FP-1 and KAN-2 RP-1),and DNA sequence of the flanking fragements were analyzed with chain termination method.The results showed that the insertion location of Tn5 transposon in genomes of mutants subcultured for 100 passages was consistent with the corresponding primary mutants,respectively,and the similarity of Tn5 transposon insertion-site flanking sequence of primary mutants and mutants subcultured for 100 passages was over 95%.From the study above,we can conclude that Tn5 transposon DNA is stably inserted into the genomic DNA of the Ralstonia solanacearum strain PRS-84.4.Biological characteristics analysis of Tn5 transposon insertion-site in mutants subcultured for 100 passegesThe mutants from wild-type strain PRS-84 mutagenized with Tn5 transposon were cultured and passaged for 100 times on NA medium and select medium containing Kanamycin.Colonial morphology of mutants during subculture was observed on Triphenyltetrazolium chloride(TTC)medium,respectively.The result showed that during continuous passage culture,there was no significant change in colonial morphology of most mutants,while there was a little change in colonial morphology(colony size,color,red center and white edge)of a few mutants.The cells from mutants PRS-84-6-13 and PRS-84-10-69 were cultured in NA medium,respectively.The OD values of bacterial solution from these two mutants were measured with spectrophotometer at a wavelength of 600nm,and then,the parameters of OD values were used to draw the growth curves.The result showed that there was little difference between mutant PRS-84-6-13 subcultured for 100 passages and its primary mutant in growth curves.While,the growth curve of mutant PRS-84-10-69 cultured for 100 passages exhibited a longer time in lag phase,reduced growth rate in logarithmic phase and decreased number of bacteria in stationary phase,compared with the primary mutant PRS-84-10-69.From the above results,we can conclude that the biological characteristics of mutants passaged for 100 times were relatively stable.