Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori.
In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/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(2)/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(2)/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.
Michielse
C B
CB
Institute of Biology, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.
Arentshorst
M
M
Ram
A F J
AF
van den Hondel
C A M J J
CA
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001230:Aspergillus / Q000235:genetics*; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D018014:Gene Transfer Techniques; D005800:Genes, Fungal; D005819:Genetic Markers; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005819:Genetic Markers