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Agrobacterium tumefaciens-mediated transformation(ATMT) is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants in the filamentous fungus,Colletotrichum gloeosporioides,by ATMT insertion of a trapping vector(pCAHPH) that carries a promoterless hygromycin phosphotransferase(hph) gene. Transformants were selected on the media containing 200 μg/mL hygromycin B,and screened for pathogenicity-related gene mutants. Their pathogenicity-related mutants T-DNA flanking sequences were then cloned and analyzed. Hph genes were amplified from mutant genomic DNA but not from wild-type DNA,indicating that the phenotypic alternations of these mutants were the results of T-DNA insertion. T-DNA flanking sequences were obtained using modified thermal asymmetric interlaced PCR. Two right-sided flanking sequences were highly homologous to proteins from other species.
Agrobacterium tumefaciens-mediated transformation (ATMT) is an efficient tool for insertional mutagenesis and is used in a wide variety of plants. This paper reports a promoter trapping method to generate mutants (pCAHPH) that carries a promoterless hygromycin phosphotransferase (hph) gene. Transformants were selected on the media containing 200 μg / mL hygromycin B, and screened for pathogenicity-related gene mutants. Their pathogenicity-related mutants T-DNA flanking sequences were then cloned and analyzed. Hph genes were amplified from mutant genomic DNA but not from wild-type DNA, indicating that the phenotypic alternations of these mutants were the results of T-DNA insertion. T-DNA flanking sequences were obtained using modified thermal asymmetric interlaced PCR. Two right-sided flanking sequences were highly homologous to proteins from other species.