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 Ras I, Alu I and Hae HI , respectively. The results of electrophoresis showed that fragment which digested by Hae IH , ranged in size from 50 bp to 500 bp and were too short to use for SSH. In contrast, the fragments which digested by Ras I and A lu I , 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 Po82△c00283. Based on the mutant strain, the complement strain was also constructed, named Po82△c00283-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 Po82△c00283 was decreased compared with the wild type Po82 and the complement strain. The pathogenicity of the complement strain Po82△c00283-pML123-c00283 was restored to the wild-type level. Growth curve analysis indicated that Po82Ac00283 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.%植物青枯菌(Ralstonia solanacearum)致病力分化菌株Po82兼具2号小种和NPB(非香蕉致病)菌株的致病特征.为了研究该菌株致病力分化机理,本实验以Po82为待测菌株,构建了抑制性差减杂交文库.建库质量分析结果表明,具有较高的接头连接以及文库构建效率,插入片段平均长度为300 bp,文库构建质量较好.对文库中随机挑取阳性克隆进行测序,共筛选出44个Po82菌株差异基因.生物信息学分析结果表明,所得差异基因主要涉及新陈代谢、转座酶、膜结构、分泌蛋白、毒力、转录因子以及未知功能等.利用基因敲除技术对其中的c00283基因功能进行了研究,结果表明,该基因在Po82菌株致病过程中起重要作用.半定量RT-PCR结果表明,c00283基因在hrp诱导培养基中的表达情况与hrpB基因一致,初步确定其为Ⅲ型分泌系统效应子.基础生物学实验结果表明,该基因对Po82菌株的生长、生物膜形成及运动性没有影响.青枯菌Po82菌株抑制性差减杂交文库的构建及致病相关基因的功能分析,为后续致病力分化的分子机理研究提供基础材料.
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