植物青枯菌aac基因突变株的构建及其致病性的测定

根据青枯菌(Ralstonia solanacearum)与群体猝灭相关aac基因序列设计PCR引物，扩增获得了aac基因，并将基因克隆到pGEM-T- easy载体中。将庆大霉素基因插入aac基因内部，使其突变，将含有庆大霉素基因的aac突变基因亚克隆进入自杀质粒pDS132中，构建成aac基因重组自杀质粒pDS-aac’-Gm。将自杀质粒电转化至青枯菌GMI 1000菌株中，通过体内同源重组，置换其野生型的aac基因。通过三步筛选法和PCR扩增鉴定，筛选获得了具有庆大霉素抗性的青枯菌aac基因突变株(GMI 1000-m)。接种番茄结果显示，青枯菌aac突变株的致病性较野生型GMI 1000明显下降。证明aac基因在青枯菌致病过程中具有重要作用。

Construction of aac Gene Mutated Strain of Ralstonia solanacearum and Its Pathogenicity Test

According to the sequence of aac gene associated with quorum-quenching of Ralstonia solanacearum, the PCR primers were designed to amplify aac gene, then the PCR fragment was cloned into pGEM-T-easy vector. The recombinant plasmid containing aac gene was mutated by introducing the gentamicin-3-acetyltransferase gene (Gmr gene). Then aac gene containing Gmr gene was subcloned into suicide vector pDS132, named pDS-aac’-Gm. Moreover, GMI 1000 strain was used to construct aac gene mutant by homologous recombination. The marked aac strain inserted with Gmr gene was selected by three-step methods and aac inserted by Gm gene on the genome was determined by PCR. pDS-aac’-Gm constructed from the sequence of R. solanacearum resulted in the construction of the in-frame insertion aac gene mutant of GMI 1001 successfully. The result of tomato inoculation test showed that the GMI 1000-m strain were much less virulent on tomato than the wild-type GMI 1000, which indicated that the aac gene is a very important factor in the pathogenesis of R. solanacearum.