ABSTRACT
A full-length cDNA coding for a putative adenosine deaminase (Fv-ada) was isolated from the basidiomycete Flammulina velutipes. Fv-ada encodes a polypeptide consisting of 537 amino acid residues, which has a consensus sequence conserved among adenosine deaminase-related growth factors (ADGF) found in several metazoa, including chordates and insects. Fv-ada transcript was detected at all stages of growth in dikaryotic F. velutipes cells, with a peak at the primordial stage. Heterologous expression of Fv-ada in the yeast Pichia pastoris produced recombinant Fv-ADA that catalyzed the conversion of adenosine to inosine. Dikaryotic mycelia from F. velutipes were transformed with the binary plasmid pFungiway-Fv-ada, which was designed to suppress the expression of Fv-ada through RNA interference. The growth rates of the resulting transformants were retarded in response to the degree of suppression, indicating that Fv-ada plays an important role in the mycelial growth of F. velutipes. These results suggested that ADGF could function as growth factors in fungi, as is seen in other eukaryotes.
Subject(s)
Adenosine Deaminase/genetics , Flammulina/genetics , Intercellular Signaling Peptides and Proteins/genetics , Adenosine Deaminase/metabolism , Adenosine Deaminase/physiology , Amino Acid Sequence , Flammulina/enzymology , Flammulina/growth & development , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/physiology , Molecular Sequence Data , Mycelium/growth & development , Pichia/genetics , Pichia/metabolism , RNA Interference , Sequence Homology, Amino AcidABSTRACT
Using fluorescence differential display, cDNAs specifically expressed at the primordial stage of fruiting body development were isolated from the basidiomycete, Flammulina velutipes. Seventy-five cDNAs were sequenced and compared with the amino-acid sequences of proteins in the database by BLASTX search. Significant similarity was found for 29 cDNAs coding for proteins with known function, GTP-binding protein, growth factor, ubiquitin-proteasome, cytochrome P450 and hydrophobin, all of which would be associated with fruiting body development. Seventeen cDNAs were not similar to proteins in the database and may represent unique genes that play specific roles in the process of fruiting in F. velutipes.
Subject(s)
Agaricales/growth & development , Fruiting Bodies, Fungal/growth & development , Fungal Proteins/genetics , Gene Expression Profiling , Gene Expression Regulation, Fungal , Agaricales/genetics , Agaricales/metabolism , Blotting, Northern , Cloning, Molecular , DNA, Complementary , Fruiting Bodies, Fungal/genetics , Fruiting Bodies, Fungal/metabolism , Fungal Proteins/metabolism , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
Hydrophobin cDNA (fv-hyd1), which is specifically expressed during fruiting body development, was isolated from the basidiomycete Flammulina velutipes by differential display screening. Analysis of the genomic structure of fv-hyd1 revealed an open reading frame (ORF) composed of 363 nucleotides and interrupted by three introns. The deduced amino acid sequence of FV-HYD1 showed a similarity to those of other fungal class I hydrophobins and contained eight cysteine residues highly conserved among hydrophobin proteins. The pattern of the hydropathy plot of FV-HYD1 was similar to those of class I hydrophobins. Southern blot analysis of genomic DNA showed that fv-hyd1 existed as a single copy. Northern blot analysis indicated that the fv-hyd1 transcript was not present in vegetative mycelia but markedly increased in level at the primordial stage. Moreover, the fv-hyd1 transcript was abundant even at the mature fruiting body stage. This result indicates that fv-hyd1 could encode a hydrophobin closely associated with fruiting body development.
