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Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes

  • Applied Genetics and Molecular Biotechnology
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Abstract

The glyceraldehyde-3-phosphate dehydrogenase gene of Flammulina velutipes was isolated. The complete gpd sequence (from ATG to TAA) was 1,489 bp in length and contained nine introns. The locations of these nine introns were similar to those of other basidiomycetes, which might reflect the evolutionary divergence of these mushrooms. The F. velutipes gpd gene was found to encode a protein of 339 amino acids and its putative amino acid sequence revealed a high similarity to an analogous protein deriving from other basidiomycetes. Results of Southern blot analysis suggested that there existed only one copy of the gpd gene in the genome of F. velutipes and that there was one typical TATA box and two CAAT boxes located in the 5′ flanking region. The F. velutipes gpd promoter was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selection marker. Using the resulting construction, hph was efficiently transformed into F. velutipes by basidiospore electroporation. No false-positive antibiotic-resistant cultures were detected by PCR amplification and the hygromycin resistance trait was maintained stably during mitotic cell division for 3 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. This rapid and convenient electroporation procedure offers new prospects for the genetic manipulation and a tool for tagging genes of this important edible mushroom species. Sequence data will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under accession number AF515622.

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Acknowledgements

The financial support of the National Science Council of the ROC (Grant No. NSC91-2317-B-002-040) is greatly appreciated. We thank Dr. Ruey-Shyang Hseu (Institute of Microbiology and Biochemistry, National Taiwan University) for his valuable scientific advice and encouragement associated with the preparation of this manuscript.

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Authors and Affiliations

  1. Institute of Microbiology and Biochemistry, National Taiwan University, No 1, Section 4, Roosevelt Road, Taipei, 106, Taiwan

    Chun-Yi Kuo, Shu-Yu Chou & Ching-Tsan Huang

  2. Department of Biochemical Science and Technology, National Taiwan University, No 1, Section 4, Roosevelt Road, Taipei, 106, Taiwan

    Ching-Tsan Huang

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  1. Chun-Yi Kuo
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  2. Shu-Yu Chou
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  3. Ching-Tsan Huang
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Correspondence to Ching-Tsan Huang.

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Kuo, CY., Chou, SY. & Huang, CT. Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes . Appl Microbiol Biotechnol 65, 593–599 (2004). https://doi.org/10.1007/s00253-004-1635-1

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  • DOI: https://doi.org/10.1007/s00253-004-1635-1

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