Identification, cloning and expression analysis of ECM33 gene in Aspergillus niger

ECM33 gene encodes a protein which belongs to a large group of GPI anchored protein family. Although the precise molecular function of this protein is unknown, several studies have demonstrated an important role in fungal cell wall integrity and virulence. The aim of this study was to identify and clone ECM33 gene in Aspergillus niger. Also the expressional analysis of the gene was carried out. The complete genomic sequence of ECM33 gene along with 5' and 3' flanking regions has been identified in Aspergillus niger by means of bioinformatics and using ECM33 gene of A. fumigatus as template. The genomic fragment was PCR-amplified using specific primers and subsequently was cloned. The expression analysis was performed by RT-PCR. The Aspergillus niger ECM33 gene containing 1327 bp was cloned as a 5.2 kb fragment. Bioinformatic analysis predicted the presence of two introns which was confirmed experimentally. The gene product is a 397 amino acids protein with approximately 70% identity to the homologue proteins from other aspergilli. Expression analysis of ECM33 under different growth conditions including different temperatures, different carbon sources, and differentnitrogen sources confirmed a constitutive expression. This is the first report on characterization of ECM33 gene in an industrially important fungus, Aspergillus niger. The cloned fragment will be used in construction of gene disruption cassette which will be subsequently applied in gene knock out experiments.