An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.
A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.
Chakraborty
B N
BN
Department of Biological Sciences, University of Calgary, Alta., Canada.
Patterson
N A
NA
Kapoor
M
M
D001203:Ascomycota / Q000235:genetics / Q000502:physiology; D001236:Aspergillus oryzae / Q000235:genetics / Q000502:physiology; D015139:Blotting, Southern; D004558:Electric Stimulation; D005658:Fungi / Q000235:genetics* / Q000502:physiology; D009492:Neurospora crassa / Q000235:genetics / Q000502:physiology; D010407:Penicillium / Q000235:genetics / Q000502:physiology; D013172:Spores, Fungal; D014170:Transformation, Genetic