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) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, 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 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA12-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.
Linnemannstöns
P
P
Institut für Botanik, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
Voss
T
T
Hedden
P
P
Gaskin
P
P
Tudzynski
B
B
D015139:Blotting, Southern; D004262:DNA Restriction Enzymes / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D017353:Gene Deletion; D005800:Genes, Fungal; D005874:Gibberella / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005875:Gibberellins / Q000096:biosynthesis* / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D014170:Transformation, Genetic
D005875:Gibberellins; D004262:DNA Restriction Enzymes