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青枯雷尔氏菌Tn5转座子无致病力突变株构建及其生物学特性
程本亮1,车建美 2,刘波3
1.
2. 福建省农业科学院农业生物资源研究所
3. 福建省农业科学院生物技术研究所
Construction of Avirulent Mutants of Ralstonia solanacearum by Tn5 Transposon and Analysis of Its Biological Characteristics
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摘要 青枯雷尔氏菌(Ralstonia solanacearum)是植物细菌性青枯病的病原菌,为研制植物疫苗菌剂提供遗传稳定且突变位点明确的青枯雷尔氏菌无致病力菌株,本研究利用EZ-Tn5转座子随机插入青枯雷尔氏菌(Rs91)构建青枯雷尔氏菌无致病力突变体库,通过电击转化,筛选获得13株具有无致病力菌株形态的突变株。研究了其中5株突变株的插入位点和生物学特性,结果表明,其插入位点分别位于phcA和pchS基因,其生长速率和相对胞外多糖含量显著低于Rs91,而最适pH值和温度未改变。其发酵液经分光光度计扫描,结果显示,突变株在同一波长下的光吸收值均大于Rs91,经聚类分析,显示它们与Rs91可聚为不同的3类,即Rs91为第Ⅰ类,phcA基因突变株为第Ⅱ类,phcS基因突变株为第Ⅲ类。经番茄(Lycopersicon esculentum)盆栽苗致病力检测,15 d后均未发病,确定为无致病力青枯雷尔氏菌。本研究为研制防治青枯病的植物疫苗菌剂提供了基础资料。
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程本亮
车建美
刘波
关键词 青枯雷尔氏菌TTC培养基无致病力突变株反向PCR胞外多糖    
Abstract:Ralstonia solanacearum is the agent of bacterial wilt. In order to provide the development of plant vaccine with avirulent mutants of R.solanacearum whose genetics are stable and insetion sites, this study constructed the avirulent mutants of R.solanacearum(strain Rs91) using EZ-Tn5 transposon mutagenesis. Through electroporation and screening, thriteen avirulent mutants were obtained. Results showed that the insertion sites of the 5 mutants were found to locate in phcA and phcS genes separately. The growth curves and the relative contents of Exopolysaccharide (EPSⅠ) of the 5 mutants were significantly lower than that of the original strain Rs91, but their optimum temperature and pH for the mutants to grow changed a few. The supernatants of the 5 mutants cultured in the TTC liquid medium were scanned by the UV-spectrophotometer, and the result showed that the absorbance of the mutants was higher than that of Rs91. The result of scanning was analyzed by UPGMA dendrogram. The cluster was displayed that the 5 avirulent mutants and Rs91 could be separated into 3 groups, e.g. the original strain Rs91 was in groupⅠ, the mutants of phcA- were in groupⅡ, and the mutant of phcS- was in groupⅢ. The 5 mutants were determined by the bioassay of potted tomato (Lycopersicon esculentum) and they did not fall ill after 15 days. The 5 mutants were determined to be avirulent R.solanacearum. This study provides foundation data for developing plant vaccine against bacterial wilt.
Key wordsRalstonia solanacearum    TTC medium    avirulence mutant    IPCR    EPSI
收稿日期: 2010-04-21     
通讯作者: 刘波   
引用本文:   
程本亮1,车建美 2,刘波3. 青枯雷尔氏菌Tn5转座子无致病力突变株构建及其生物学特性[J]. , 2011, 19(1): 26-35.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2011/V19/I1/26
 
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