农业生物技术学报2012，Vol.20Issue(7)：745-755,11.DOI:10.3969/j.issn.1674-7968.2012.07.004
应用抑制性差减杂交法筛选植物青枯菌致病相关基因
Screening for Pathogenicity-related Genes in Ralstonia solanacearum by Suppression-subtractive Hybridization
摘要
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 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.关键词
青枯菌/抑制性差减杂交/Ⅲ型分泌系统效应子/致病性Key words
Ralstonia solanacearum/ Suppressive subtractive hybridization/ Type Ⅲ secretion system effector/ Pathogenicity引用本文复制引用
刘蕾,徐进,许景升,张丽勍,张昊,冯洁..应用抑制性差减杂交法筛选植物青枯菌致病相关基因[J].农业生物技术学报,2012,20(7):745-755,11.基金项目
国家重点基础研究发展计划(973)项目(No.2009CB119200)、国家高技术研究与发展计划(863)项目(No.2012AA101501)和国家自然科学基金资助项目(No.31101409) （973）
