Comparison of different transformation methods for Aspergillus giganteus
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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 108 protoplasts/µg 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.
Keywords.Protoplast transformation Electroporation Biolistic transformation Agrobacterium tumefaciens-mediated transformation
We would like to thank Susanne Engelhardt and Barbara Walewska for technical assistance. We thank also Cees van den Hondel and Paul Hooykaas for providing us with the plasmids pAN7-1 and pUR5750, respectively. We are grateful to Caroline Michielse for helpful suggestions regarding A. tumefaciens-mediated transformation. The work was carried out in compliance with the current German laws governing genetic experimentation.
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