| Ralstonia solanacearum is one kind of soil-borne plant pathogen of bacterial wilt, Gram-negative plant pathogenic bacterium. The pathogen is widely distributed throughout the world and can infect more than hundreds plant species belonging to over 40 families. It seriously affected crop yield, caused enormous losses to agricultural production.In this study, Tn5 insertion mutant library of R.solanacearum strains EP-1 was constructed, and 15 EPS significantly reduced mutants from 24,500 clones were screened. The complete DNA sequences of the genes disrupted by Tn5 transposon were obtained by hi TAIL-PCR. The extracellular enzyme activity, biofilm and motion detection,and the pathogenicity of those mutants were characterized. By using q RT-PCR to analysis differences related gene expression, which will be benefit for further study of R. solanacearum pathogenic and its regulation to build rapid identification of R. solanacearum virulence. The results are as follows:1. The Tn5 transposon random insertion library of R. solanacearum strain EP-1 was constructed.. Among of which, 15 EPS significantly reduced mutant were screened, named Em2, Em3, Em4, Em28, Em29, Em31, Em32, Em36, Em37, Em51, Em56, Em58, Em60, Em61 and Em64. Meanwhile, R. solanacearum strains EP-1, GMI1000, TM-1 and PN-1 were subcultured to the 60 th generation(EP-60), the 25 th generation(GMI1000-25), and the 30 th generation(TM-30 and PN-30)2. The complete DNA sequence of the genes disrupted by Tn5 insertion were obtained by hi TAIL-PCR. Blast analysis showed that the mutant Em2, Em3, Em4, Em29, Em31 and Em37 was mutated in the same gene phc A. This gene has 1,044 bp, encoding 347 amino acids, is the globle transcriptional regulator; Em28 was mutated in gene eps B. The gene has 2,256 bp, encoding 751 amino acids, is putative tyrosine-protein kinase eps B and related to the biosynthesis of EPS; Em32 was mutated in gene CMR15-30160. The gene has 690 bp, encoding 229 amino acids, is a conserved protein with unknown function; Em36 was mutated in gene gst O. The gene has 690 bp, encoding 229 amino acids, is a probable glutathione-s-transferase protein; Em51 was mutated in gene pst S2. The gene has 1,062 bp, encoding 353 amino acids, is a probable phosphate-binding periplasmic precursor(pbp) ABC transporter protein; Em56 was mutated in gene asd. The gene has 1,104 bp, encoding 367 amino acids, is a probable aspartate-semialdehyde dehydrogenase oxidoreductase protein; Em58 was mutated in gene RSc1351. The gene has 1,245 bp, encoding 414 amino acids, is a putative transmembrane sensor histidine kinase transcription regulator protein; Em60 was mutated in gene RSc1584. The gene has 441 bp, encoding 146 amino acids, is a putative transcription regulator protein; Em61 was mutated in gene RSc1620. The gene has 243 bp, encoding 80 amino acids, is a probable transmembrane potein; Em64 was mutated in gene pck G. The gene has 1,869 bp, encoding 622 amino acids, is a probable phosphoenolpyruvate carboxykinase protein.3. q RT-PCR analysis showed that the related genes of EPS biosynthesis and quorum sensing in those mutants were significantly down-regulated. Inoculating the host plants demonstrated that the phathogenicity of these mutants were decreased, and the motility and formation of the biofilm were also affected.4. The detection method of R. solanacearum pathogenicity differiation was initially established. Based on the analysis results of those EPS biosynthesis defective mutants, the genes phc A, gst O, RSc1351, and RSc1584 could be chosed as the targets, the pathogenicity of R. solanacearum strains could be evaluated according to their transcription level. |
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