The Gibberella fujikuroi niaD Gene Encoding Nitrate Reductase: Isolation, Sequence, Homologous Transformation and Electrophoretic Karyotype Location

Abstract

The Gibberella fujikuroi niaD gene, encoding nitrate reductase, has been isolated and used to develop an efficient homologous transformation system. A cosmid vector designated pGFniaD was generated based on niaD selection and shown to give comparable transformation efficiencies. Using pGFniaD, a genomic library was prepared and used for genetic transformations, giving frequencies of up to 200 transformants per μg DNA. Of 15 transformants analysed by Southern blots, six showed homologous integration whilst the remaining nine integrated at heterologous sites, indicating that the vector may be used reliably for both types of integration. The system therefore may be used both for self-cloning of gibberellin biosynthetic genes on the basis of complementation of defective mutants, and also for gene disruption experiments. Electrophoretic karyotype determination suggested at least 11 chromosomes ranging from 2 to 6 Mb, the total genome size being at least 37 Mb. The niaD gene was assigned to chromosome V by Southern blot analysis. The niaD gene is interrupted by one intron, and remarkably the promoter sequence, but not the 3′ untranslated sequence, is highly homologous to that of the corresponding Fusarium oxysporum gene. This situation appears to be unique with respect to the promoter regions of corresponding genes in related species of filamentous fungi.