Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori
article
In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl<sub>2</sub>/PEG-mediated transformation was compared. For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl<sub>2</sub>/PEG protoplast transformation. For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl<sub>2</sub>/PEG protoplast transformation. Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55%. Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement. © 2004 Elsevier Inc. All rights reserved.
Topics
BiotechnologyAgrobacterium tumefaciensAspergillus awamoriDouble marker enrichment techniqueFilamentous fungiGene targetingCalcium chlorideMacrogolAspergillus awamoriDNA flanking regionFungal geneFungal geneticsGene cassetteGene deletionGene frequencyGenetic transformationHomologous recombinationNonhumanNucleotide sequencePriority journalProtoplastRhizobiumAspergillusDNA, FungalGene Transfer TechniquesGenes, FungalGenetic MarkersMolecular Sequence DataRecombination, GeneticRhizobium radiobacterSequence Analysis, DNATransformation, GeneticAgrobacteriumAgrobacterium tumefaciensAspergillusAspergillus awamoriFungi
TNO Identifier
43043
Source
Fungal Genetics and Biology, 42, pp. 9-19.
Pages
9-19
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