Cloning and nucleotide sequence of a cDNA encoding the antifungal-protein ofAspergillus giganteusand preliminary characterization of the native gene

A.giganteus secretes a 5.8 kDa protein which inhibits the growth of a variety of fungal species (1). In order to clone the cDNA encoding the antifungal-protein, a cDNA expression library was constructed in XZAP (Stratagene) using polyARNA from induced mycelia as template for cDNA synthesis. A complete cDNA coding for the antifungal-protein was isolated by immunoscreening of the XZAP library with polyclonal antibodies. The cloned cDNA was used as a hybridization probe for the isolation of the native antifungal-protein gene from a XEMBL3 library of A. giganteus. Sequencing of the antifungal-protein cDNA revealed an open reading frame (ORF) of 177 nucleotides (Fig. 1). The amino acid sequence deduced from the major part (nucleotide 28—177) of the ORF corresponds exactly to the entire sequence determined for the secreted protein (1). Additionally there is an N-terminal leader sequence of 9 amino acids which might be responsible for secretion. The functional identity of the leader peptide is currently under investigation using the transformation system recently established for A. giganteus (2). Northern analysis of total RNA from 24 and 48 h old mycelia cultivated in medium containing either starch or glucose as major carbon source indicates, that expression of the antifungal-protein gene is inhibited in the presence of glucose. While the amount of antifungal-protein mRNA increases with time in the presence of starch, only a weak signal was obtained with RNA from mycelia grown in glucose containing medium after 24 h (Fig. 2a). The apparent size of the antifungal-protein mRNA is around 520 bp which suggests that the ORF is flanked by relatively long untranslated regions. Unique restriction fragments of genomic DNA of 2.7 kb (£coRI) and 13.5 kb (BamHI) hybridizing to the cDNA probe could be identified by Southern analysis (Fig. 2b). A restriction map of the 2.7 kb fcoRI fragment cloned from the XEMBL3 library shows the position of the antifungal-protein gene within this genomic region (Fig. 3). Preliminary sequencing data indicate the presence of a 55 bp long intron which is located 122 bp downstream of the beginning of the ORF.