THE GIBBERELLA-FUJIKUROI NIAD GENE ENCODING NITRATE REDUCTASE - ISOLATION, SEQUENCE, HOMOLOGOUS TRANSFORMATION AND ELECTROPHORETIC KARYOTYPE LOCATION

The Gibberella fujikuroi niaD gene, encoding nitrate reductase, has be en isolated and used to develop an efficient homologous transformation system. A cosmid vector designated pGFniaD was generated based on nia D selection and shown to give comparable transformation efficiencies. Using pGFniaD, a genomic library was prepared and used for genetic tra nsformations, giving frequencies of up to 200 transformants per mu g D NA. Of 15 transformants analysed by Southern blots, six showed homolog ous integration whilst the remaining nine integrated at heterologous s ites, indicating that the vector may be used reliably for both types o f integration. The system therefore may be used both for self-cloning of gibberellin biosynthetic genes on the basis of complementation of d efective mutants, and also for gene disruption experiments. Electropho retic karyotype determination suggested at least 11 chromosomes rangin g 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 sequen ce, 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 gen es in related species of filamentous fungi.