Establishment And Optimization Of Tn5 Transposon Mutagenesis System In Ralstonia Solanacearum Strains Isolated From Infected Pogostemon Cablin Benth.

Pogostemon cablin(Blanco)Benth.is very sensitive to bacterial wilt,a devastating disease caused by Ralstonia solanacearum.R.solanacearum strains(PRS-84,PRS-1 and PRS-58)used in this study were isolated from diseased P.cablin in our previous study.Strain GIM1.70,obtained from Guangdong Institute of Microbiology,was isolated from infected tomato plants.Tn5 Transposome was used as a mutagenic tool.It consists of Tn5 transposase and Tn5 transposon DNA that contains a kanamycin resistance gene.The competent cells were prepared and mutagenized with Tn5 transposon by electroporation.Then,kanamycin-resistant(Kanr)colonies were selected on medium with kanamycin and positively identified by PCR using primers based on Kan gene.Factors influencing electrotransformation efficiency were investigated.Further,growth characteristics of 18 selected transformants and transposon insertion stability in these mutants were analysed respectively.A practical Tn5 transposon mutagenesis system for R.solanacearum isolated from P.cablih was established.The effective system will help to create mutants of R.solanacearum strain which may be used to discover and develop potential biocontrol agents against bacterial wilt.1 Review on relevant Iiteratures in domestic and abroadThe present studies on germplasm resources,cultivation techniques and disease control of Pogostemon cablin were summarized.The researches on biological control progress of bacterial wilt disease were reviewed.Besides,researches on transposon insertion technology,Tn5 and its use in mutagenesis of bacteria against plant diseases were summarized as well.2 Factors affecting transformation efficiency of Tn5 transposon mutagenesis in Ralstonia solanacearum strainsRalstonia solanacearum bacterial cells were cultured on medium containing kanamycin at different levels,in order to detemine the sensitivity of the cells to the antibiotic.Kanamycin at 10 μg/mL,almost no cells could grow on the medium.Thus 10 μg/mL kanamycin was used as selection pressure for the selection of transformed cells.The competent cells were prepared and eletroporated with Tn5 transposon.The effects of.various parameters on electrotransformation efficiency of strain PRS-84 were investigated.The parameters included kanamycin selection pressure,cell growth phase,cell density and electric field strength.A great transformation efficiency with around 3×104 CFU/μg DNA was obtained under the optimized parameters as followed,1×1010 CFU/mL of the competent cells harvesting at OD600 0.250 of 10-fold dilution were mixed with Tn5 transposon and then electroporated at a field strength of 12.5 kV/cm.Furthermore,differences on transformation efficiency among various strains were investigated as well.Among the four strains,maximum transformation efficiency was obtained in strain GIM1.70 with 1389.65 ×104CFU/μg DNA,which was over 500-fold higher than those in other three strains.The transformation efficiency.of the strains from P.cabl in was similar.3 Analysis on growth characteristics of mutants from Ralstonia solanacearumDifferences in colonial morphology between wild-type strain PRS-84 and its mutant strains were observed on Triphenyltetrazolium chloride(TTC)medium.No variation in colonial morphology of most mutants was observed.However,there was a little difference in’ colonial morphology between a few mutants and the wild-type strain.Growth curves of cells from mutant strains(PRS-84-10-43 and PRS-84-9-103)and the wild type strain were measured as absorbance at 600 nm.Results indicated that growth curves were almost the same between the two tested mutants and the wild-type strain.4 Analysis on genetic stabil ity of mutants from Ralstonia solanacearumDNA extracted from primary cultures and subcultures after 5,10,15,20 and 25 passages respectively of mutant strains was amplified for Kanrgene fragment by PCR.Results demonstrated that Kanr gene exists in genomic DNA of the cells from mutant subcultures.There was almost no difference in colonial morphology of most mutants between their primary cultures and subcultures after 25 serial passages.Growth curves were similar between primary cultures and the twenty-fifth passage subcultures from two tested mutants(PRS-84-10-43 and PRS-84-9-103),while cell number of the subcultures was a little smaller in the same growth phase.