Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme-mediated integration and conventional transformation-mediated mutagenesis

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) bi osynthesis by transformation-mediated mutagenesis:with the vector pAN7-1. W e recovered 24 GA-defective mutants in one of nine transformation experimen ts performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector i nto the same site. The addition of a restriction enzyme by restriction enzy me-mediated integration (REMI) significantly increased the transformation r ate and the rate of single-copy integration events. Of 1 ,600 REMI transfor mants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conve ntionally transformed GA-deficient mutants. Biochemical analysis; of the tw o REMI mutants confirmed that they cannot produce ent-kaurene, the first sp ecific intermediate of the GB pathway. Feeding the radioactively labelled p recursors ent-kaurene and GA(12)-aldehyde followed by high-performance liqu id chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. So uthern blot analysis and pulsed-field gel electrophoresis of the transforma nts using the bifunctional ent-copalyl diphosphatelent-kaurene synthase gen e (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants o btained by conventional insertional transformation. We conclude that transf ormation procedures with and without the addition of restriction enzymes ca n lead to insertion-mediated mutations and to deletions and chromosome tran slocations.