水稻基腐细菌Tn5插入突变体库的构建及其致病相关突变体的筛选

采用转座子Tn5插入突变技术,共获得5 261个接合子。通过马铃薯软腐快速筛选和PCR检测验证,获得267个使马铃薯软腐能力丧失或下降的Tn5插入突变体,并测定其在水稻上的致病力和烟草上的过敏性坏死反应(HR)。结果表明:在267个突变体菌株中,对水稻无致病力的有71个,其中在烟草上无HR反应的有59个,产生HR反应的有12个;对水稻致病力下降的有68个,其余突变体菌株对水稻致病力与野生菌株差异不明显。另外,267个突变体菌株中有146个无HR反应,其中有90个对水稻无致病力或者致病力下降。致病力的分析结果表明,获得的突变体是由Tn5插入Dickeya zeae染色体上不同的致病相关基因位点导致的。

Construction of a Tn5-inserted mutant library of Dickeya zeae and screening pathogenicity-related mutants

In this report,5 261 transconjugants of Dickeya zeaewere obtained by a mini-Tn5 transposon.PCR determination revealed that 267 mutants were inserted by Tn5.Pathogenicity assay on potato indicated that they all lost or reduced the ability to cause soft rot on potato.Furthermore,the assays for pathogenicity or virulence on rice and hypersensitive reaction(HR) on tobacco showed that 71 mutants had no virulence on rice,of which 59 mutants had no HR on tobacco.In addition,68 mutants reduced virulence in rice,the rest of mutants have no obvious difference with EC1.Moreover,146 of 267 Tn5 mutants had no HR on tobacco,of which 90 had no or reduced the virulence on rice.The above results indicated that the mutants were produced by inserting Tn5 transposon into different genes in D.zeae chromosome.The screened mutants from our Tn5-inserted mutant library provide the basis for the further understanding of molecular mechanisms of D.zeae pathogenicity in rice.