Background and Aim: Annexins are a large family of calcium-phospholipid binding proteins which are distributed among nearly all eukaryotes. These proteins have been found in a wide range of fungi including ascomycetes, basidiomycetes and oomycetes. The aim of present study has been the investigation of cellular localization of ANXC3.1 in filamentous fungus Aspergillus niger (A. niger). The effect of ANXC3.1 overexpression on growth rate and protein secretion of A.
niger has also been investigated.
Materials and Methods: In this experimental study, the annexin C3.1 gene was PCR-amplified using genomic DNA of Aspergillus niger as a template. Following the confirmation of gene sequence, it was used in the preparation of two inducible gene constructs. In the first construct, annexinC3.1 was cloned in PGEM–Egfp expression vector as a GFP fusion and under the control of glucoamylase promoter. In the second construct, the gene was cloned in pMJB104 expression vector, driven by cbhB (cellobiohydrolase B) promoter. These constructs, then were transformed into A.
niger N402 using a standard method. Overexpression of each construct was induced by an inducer carbon source and the effect of annexinC3.1 overexpression on radial growth rate and protein secretion was analysed in transformants. Comparisons of radial growth rates, as well as protein secretion level, between wild type and transformants was performed using t test. Cellular localization of annexinC3.1 was investigated in annexin-GFP expressing transformant by fluorescent microscopy.
Results: The effect of annexinC3.1 overexpression on growth rate and protein secretion was investigated in Aspergillus niger. No significance difference was observed in growth rate or level of protein secretion when transformants and wild type were compared (
α <0.05). Microscopic examination of annexin C3.1-GFP fusion protein demonstrated that annexinC3.1 is a cytosolic protein which is distributed along the fungal mycelium.
Conclusion: This is the first report on annexinC3.1 cellular localization. Annexin C3.1 is a cytosolic protein and its overexpression in A. niger does not increase the protein secretion level.
