Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus

Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl 2-mediated transformation of protoplasts. Electroporation yield...

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Bibliographic Details
Published in:Journal of microbiological methods 2007-09, Vol.70 (3), p.519-527
Main Authors: Marchand, G., Fortier, E., Neveu, B., Bolduc, S., Belzile, F., Bélanger, R.R.
Format: Article
Language:English
Subjects:
Agrobacterium
Agrobacterium tumefaciens
Agrobacterium tumefaciens - genetics
Basidiomycota - genetics
Basidiomycota - physiology
Biological and medical sciences
Biotechnology
Electroporation
Electroporation - methods
Fundamental and applied biological sciences. Psychology
Gene transfer
Genetic engineering
Genetic technics
Genetic transformation
Methods. Procedures. Technologies
Microbiology
Mycological methods and techniques used in mycology
Mycology
Pseudozyma antarctica
Spores, Fungal
Synthetic digonucleotides and genes. Sequencing
Transformation, Genetic
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Summary:Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl 2-mediated transformation of protoplasts. Electroporation yielded 100–200 transformants per μg of DNA per 10 8 cells after 3 days on selective medium. For its part, ATMT yielded 60–160 transformants per 10 6 input cfu after 5–10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl 2-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium.
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2007.06.014