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Abstract 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.
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Received: 17 January 2008
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