Agrobacterium tumefaciens-mediated transformation: an efficient tool for gene replacement in Aspergillus awamori.

C.B. Michielse, A.H.A. van Dijk, M. Arentshorst, A.F.J. Ram, P.J.J. Hooykaas, C.A.M.J.J. van den Hondel.

Abstract: 

Gene disruption or gene replacement is often used to generate precise deletion mutants in order to assess a possible function to the deleted gene. A. tumefaciens, a plant pathogen, which is being used for the transformation of plants, yeast and filamentous fungi, was shown to be an efficient tool for gene targeting in Kluveromyces lactis (Bundock et al., 1999). To determine the efficiency of gene replacement in Agrobacterium-mediated transformation of A. awamori, a systematic study was performed. The hygromycin selection marker was flanked with promoter and terminator sequence homologous to pyrG of varies sizes (1000 to 50 bp). Homologous recombination frequencies were determined and compared to frequencies obtained with the PEG/CaCl2 transformation method. Homologous recombination frequencies with the Agrobacterium-system increased 6-fold compared to the conventional method with 1000 bp flanks (30% versus 5%, respectively). Shortening the flanks to 500 and 250 bp led to a decrease in recombination frequencies to 5 and 1%, but these frequencies were again higher compared to the conventional method. By altering the length of the left and right flanking regions, it was shown that a long left flanking region increases the percentage of homologous recombination. Based on these data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the left border of the T-DNA plays an important role in homology search and might serve as a starting point for integration. Bundock et al. (1999), T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis, MGG 261(1): 115-21.
2003

abstract No: 

Fungal Genet. Newsl. 50 (Supl):abstract

Full conference title: 

22nd Fungal Genetics Conference
    • Fungal Genetics Conference 22nd (2001)