成团泛菌对玉米自交系PS056致病性基因yhfK的克隆

摘要

[Objective]The purpose of this study was to clone and identify a pathogenicity gene of Pantoea agglomerans causing bacterial dry stalk rot on maize.[Method]A transposon mutant library was constructed in P agglomerans by Tn5 transposon mutagenesis.The mutants with defective pathogenicity were screened using the stalk-injecting inoculation on the susceptible maize inbred line PS056.One mutant lost pathogenicity was obtained and designated as PA121.[Result]Southern blotting analysis indicated that the mutant PA121 had a single-copy Tn5 transposon which inserted into the yhfK at the site of 1 082 bp upstream.The yhfK encodes a conserved inner membrane protein, identified by bi-directional sequencing based on Tn5 sequence.Phenotypic analysis indicated a complementary plasmid retained the pathogenicity on host PS056.The activities of extraceilular enzymes, such as protease, amylase and cellulase, were not affected in mutant PAl21.RT-PCR analysis demonstrated that the yhfK regulated the transcription of hrpA.[Conclusion]This study demonstrates that the yhfK affects pathogenicity of P agglomerans,which is a critical element in the pathogenesis of P agglomerans causing bacterial dry stalk rot on maize.%[目的]通过克隆和鉴定成团泛菌(Pantoea agglomerans)的致病相关基因,阐明该种土壤习居细菌在玉米自交系PS056上引起细菌性干茎腐病的原因.[方法]利用转座子Tn5的随机插入突变特点,构建致病性菌株P.agglomerans XJ1突变体库;经对Tn5插入位点的侧翼扩增,确认突变体PA121的致病力相关基因;通过基因互补试验,证明yhfK与致病力的关系.[结果]通过对突变菌株的接种筛选,获得11个致病力丧失的突变体.Southern杂交和PCR扩增结果表明,Tn5在突变体PA121中为单拷贝插入,插入位点为细菌编码内膜蛋白的yhfK(Tn5插入在yhfK的1 082 bp处).将yhfk克隆到质粒pBBRIMCS上,并导入突变体PA121中进行互补,生物测定结果显示转化子完全恢复了正常的细菌生理功能,并能够在玉米自交系PS056上恢复致病力.RT-PCR试验证明,yhfK调控hrpA的转录.[结论]成团泛菌XJ1中yhfk是调控其对植物致病性的基因之一,具有在玉米自交系PS056上引起细菌性干茎腐病的功能.