烟草青枯菌Tn5突变体库的构建及突变位点分析

Construction of Tn5 Mutant Library of Ralstonia solanacearum and Analysis of Mutation Sites

为了从烟草青枯菌全基因组范围内发掘致病性相关基因,采用电转化法构建了一个EzTn5转座子介导的烟草青枯菌TXLLJ14-3菌株插入突变体库,该突变体库包含1.2万个突变子,经烟草上的致病性检测,共得到216个无致病力或弱致病力突变菌株。利用TAIL-PCR扩增获得其中15个无致病力烟草青枯菌的Tn5侧翼序列,并对其插入位点进行了分析,结果表明,15个突变菌株的插入位点分别位于核苷酸水解酶、糖基转移酶、转座酶和合成酶等具有不同功能的基因上,这些基因受到干扰或破坏后,可能会抑制致病相关物质的表达或分泌,或者诱导烟草对病原菌产生抗性,从而表现出无致病力特征。

In order to explore the pathogenic genes from the whole genome range of Ralstonia solanacearum,this study constructed a Ez-Tn5 transposon insertion mutant library of R. solanacearum TXLLJ14-3 by electroporation method. The library contained 12 000 mutants. There were 216 non or weak pathogenic mutant strains by the pathogenic detection on tobacco. Tn5 flanking sequences of 15 non pathogenic mutant strains were amplified by TAIL-PCR,and simultaneously,the insertion sites were analyzed.The results showed that the insertion sites of the 15 mutant strains were located at genes with different functions such as nucleotide hydrolases,glycosyltransferases,transposase and synthase. When they were disrupted or destroyed,the expression or secretion of virulence related substances might be inhibited,or tobacco resistance to pathogen be induced,thus showing no pathogenic characteristics.