Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes.
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.
Kuo
Chun-Yi
CY
Institute of Microbiology and Biochemistry, National Taiwan University, No 1, Section 4, Roosevelt Road, 106, Taipei.
Chou
Shu-Yu
SY
Huang
Ching-Tsan
CT
D024506:5' Flanking Region; D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D025141:Drug Resistance, Fungal / Q000235:genetics; D018274:Electroporation; D029968:Escherichia coli Proteins / Q000235:genetics; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D016385:TATA Box; D014170:Transformation, Genetic
D004271:DNA, Fungal; D029968:Escherichia coli Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase