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Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
Published on: March 22, 2016
T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable
Mubashir Hanif1, Alejandro Guillermo Pardo, Markus Gorfer
1Department of Biosciences, University of Helsinki, Finland. hanif@mappi.helsinki.fi
Abstract:
The T-DNA of Agrobacterium tumefaciens can be transferred to plants, yeasts, fungi and human cells. Using this system, dikaryotic mycelium of the ectomycorrhizal fungus Suillus bovinus was transformed with recombinant hygromycin B phosphotransferase (hph)and enhanced green fluorescent protein (EGFP) genes fused with a heterologous fungal promoter and CaMV35S terminator. Transformation resulted in hygromycin B-resistant clones, which were mitotically stable. Putative transformants were analysed for the presence of hph and EGFP genes by PCR and Southern analysis. The latter analysis proved both multiple- and single-copy integrations of the genes in the S. bovinus genome. A. tumeficiens transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for S. bovinus.
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