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Abstract Construction of a mutant library is a fundamental approach to new genes and functional genomics of a plant pathogen. To rapidly and accurately identify a Tn5 insertion position in a corresponding gene in Xanthomonas oryzae pv. oryzicola, the critical pathogen of bacterial leaf streak in rice, thermal asymmetric interlaced PCR (TAIL-PCR) and Tn5 transposon rescue were adopted to verify 8 virulence-reduced mutants screened from the Tn5 inserted library of the pathogen based on virulence assay in rice. TAIL-PCR revealed that 5 virulence-reduced mutants were due to that the Tn5 transposon was inserted in the general secretion protein F (gspF), cAMP-regulatory protein, integral membrane protease subunit, S-adenosylmethionine decarboxylase proenzyme, and gpsJ genes, respectively. Tn5 transposon rescue indicated that the genes in other three mutants were inserted by Tn5 were pathogencity protein, conserved hypothetical protein, and flagellum-specific ATP synthase genes, respectively. Homology analysis demonstrated that eight genes inserted by Tn5 were 100% identities to the corresponding genes in the genome sequence of the strain BLS256. The results above suggest that TAIL-PCR and Tn5 transposon rescue are effective tools for identifying Tn5 insertion position of a target gene of a plant pathogen and for isolating the target and can be used coordinatively and cooperatively.
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Received: 08 December 2008
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