Comparison of different transformation methods for Aspergillus giganteus.
Four different transformation methods were tested and compared in an attempt to facilitate the genetic transformation of Aspergillus giganteus, the producer of an antifungal protein (AFP). The fungus was transformed to hygromycin B resistance, using the hph gene of Escherichia coli by protoplast transformation, electroporation, biolistic transformation, and Agrobacterium tumefaciens-mediated transformation. Electroporation and biolistic transformation were found to be inappropriate for transforming A. giganteus, due to a low transformation yield. The conventional transformation technique based on protoplasts yielded up to 55 transformants in 10(8) protoplasts/microg DNA and was enhanced to 140-fold by A. tumefaciens-mediated transfer of its T-DNA. Here, the germination time prior to cocultivation and the fungus:bacterium ratio were found to alter the transformation efficiency. Southern blot analysis revealed that the A. giganteus transformants contained a randomly integrated single T-DNA copy, whereas multiple integration events were frequent in transformants obtained by the protoplast method.
Meyer
Vera
V
Fachgebiet Mikrobiologie und Genetik, Institut für Biotechnologie, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany. v.meyer@lb.tu-berlin.de
Mueller
Dirk
D
Strowig
Till
T
Stahl
Ulf
U
D001230:Aspergillus / Q000235:genetics*; D019470:Biolistics; D015139:Blotting, Southern; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation; D018014:Gene Transfer Techniques; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; C036483:T-DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase