Construction Of A TN5-Tagged Mutant Library And Screening Of Bismerthiazol Sensitivity Mutants In Xanthomonas Oryzae PV.Oryzae

Bacterial blight of rice which caused by Xanthomonas oryzae pv. oryzae was one of the most serious bacterial diseases of rice. Bismerthiazol [N, N-methylene-bis (2-amino-5-mercapto-1,3,4-thiadiazole] was widely used in rice bacterial blight diseases.Due to bismerthiazol having broad spectrum antibacterial activity, low toxicity, selectivity, etc features, so was long-term single-use, however, in recent years, bismerthiazol also appeared in the production of the corresponding resistance problems that bacterial diseases prevention and control of chemical faced a new challenge. Given the current still do not understand the role and resistance mechanism of bismerthiazol, the paper by constructing Tn5 random insertion mutant library, used bismerthiazol screening susceptibility changes mutants for the study of the mechanism and resistance mechanism of bismerthiazol provides a reliable test materials.The mutant library contained 16,000 mutants.2 randomly selected mutants after 25 generations in the activation, verificaed by Southern blot hybridization, the insertion point change was not found that keeping mutations good stability; 26 randomly selected mutants, Southern blot hybridization showed insertion point is random, There were single Tn5 insertion and double insertion; obtain 43 mutants Tn5 flanking sequences by RATE-PCR, making Tn5 insertion location map on Xanthomonas bacteria genome-wide, uniform distribution observed transposon Tn5 in the whole genome, the mutant library had a good saturation. Validated, the mutant library, random stability, saturation were good, library of mutants was a high quality.Experimental Determination of the bismerthiazol to the wild-type strain ZJ173 minimum inhibitory concentration MIC 70?g/ml, as a reference, by the addition of pharmaceutical in NA plates and observed colonies growth to screen Xanthomonas mutant library. Get 7 more sensitive mutants and different levels of resistance to bismerthiazol 8 mutants. For virulence and bismerthiazol susceptibility biology phenotype determined, found varying degrees of decline of 15 mutant virulence bismerthiazol sensitive and resistant; bismerthiazol to the wild-type strain ZJ173 effective concentration EC50 is 12.5?g/ml, resistant mutants highest EC5024.1?g/ml, sensitive mutant minimum EC50 3.7?g/ml. RATE-PCR Tn5 flanking sequences shows that the insertion of 8 resistant mutants were IS family (Transposase and inactivated derivatives, IS30 family), cytochrome C oxidase subunit II, single-stranded DNA-specific exonuclease and other genes,7 sensitive mutants insert signal transduction histidine kinase insert, Rhs family protein, transposon (putative transposase) and other genes.Meanwhile mutant screening,their extracellular polysaccharide content and the conductivity of the cell membrane were measured, extracellular polysaccharide found decreased levels of resistant strains; permeability of the cell membranes of all mutants were increased.