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Abstract The R. solanacearum pathogenicity variation strain Po82 has the characteristics of both strains pathogenic to banana and strains not pathogenic to banana(NPB). In order to elucidate the molecular mechanism of pathogenicity variation, the method of comparative genomics was used. Genomic DNA of the Po82 strain was compared with that of the NPB strain RUN292 by suppressive subtractive hybridization (SSH). The total genomic DNA was digested by RasⅠ, AluⅠand HaeⅢ , respectively. The results of electrophoresis showed that fragment which digested by HaeⅢ , ranged in size from 50 bp to 500 bp and were too short to use for SSH. In contrast, the fragments which digested by RasⅠ and AluⅠ, ranged in size from 500 bp to 2 000 bp, so they were appropriate to use. Detection and analysis of the library construction showed that the technique was efficient in the adaptors ligation and the subtraction. The average size of insert fragment was 300 bp. Plasmids were extracted from positive clones randomly selected from the library and then sequenced and forty-four strain-specific genes of Po82 were identified. The results of bioinformatics analysis indicated that they related to many biological processes including metabolism, transposase, membrane structure, secretion protein, virulence factor, transcription regulation and some unknown function. The mutant of c00283 gene was constructed by using homologous recombination and named Po82Δc00283. Based on the mutant strain, the complement strain was also constructed, named Po82Δc00283-pML123-c00283. The pathogenicity and the biological function of wild type, mutant strain and the complement strain were tested. Pathogenicity test showed that disease index of the mutant Po82Δc00283 was decreased compared with the wild type Po82 and the complement strain. The pathogenicity of the complement strain Po82Δc00283-pML123-c00283 was restored to the wild-type level. Growth curve analysis indicated that Po82Δc00283 mutant grew as fast as the Po82 strain in both rich medium and Boucher's minimal medium. There were no significant differences among the wild-type, mutant and the complement strain in the ability of the mobility and the biofilm formation. In this study, the SSH libraries were successfully constructed and forty-four Po82 strain specific genes were obtained. The function of one pathogenicity related gene was analyzed by using homologous recombination and other biological tests. In conclusion, the results provide basic information further elucidate the molecular mechanism of pathogenicity variation
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Received: 09 February 2012
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