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

• DOI: 10.1007/s00253-004-1635-1
• Publication Date: 2004-05-26T14:35:28Z
• Authors: Chun-Yi Kuo; Shu-Yu Chou; Ching-Tsan Huang
• Keywords: Recombination, Genetic; 0301 basic medicine; 0303 health sciences; 5' Flanking Region; Escherichia coli Proteins; Molecular Sequence Data; Gene Dosage; Glyceraldehyde-3-Phosphate Dehydrogenases; Introns; Artificial Gene Fusion; Blotting, Southern; Open Reading Frames; Phosphotransferases (Alcohol Group Acceptor); 03 medical and health sciences; Electroporation; Drug Resistance, Fungal; Gene Expression Regulation, Fungal; Amino Acid Sequence; Cloning, Molecular; Agaricales; DNA, Fungal; Promoter Regions, Genetic; Sequence Alignment

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.