earticle

영어

Cryparin is an abundant cell wall-associated hydrophobin of the chestnut blight fungus, Cryphonectria parasitica. Although cryparin is encoded as a single copy gene (Crp), it isthe most abundant protein produced by this fungus when grown in liquid culture1), 2). As the results of previous studies to characterize the transcriptional regulatory element(s) for strong expression
and hypoviral regulation, it was suggested that the fragment between nt -188 and the translation start codon appeared to be a minimal but sufficient promoter element for the efficient expression of the cryparin gene. To examine whether this small fragment is necessary and sufficient for a high level expression of the fungal gene, three different chimeric reporter genes using ORF’s of a hygromycin B resistance gene (hph), an inducible laccase (lac3) of C. parasitica and glucose oxidase (Go) of Aspergillus niger were tested3),4). When using the hphgene as a reporter gene, more than 1,000 stable transformants were obtained indicating that the 188 bp-fragment is
comparable to that of trpC promoter of A. nidulans. When using the endogenous lac3 gene as a reporter gene, which requires the presence of tannic acid forthe transcriptional induction, the lac3 transcript was observed without any induction by tannic acid. In addition, glucose oxidase as a
secretory protein was also tested for the expression. The enzymatic level of recombinant C. parasitica was within a range of two to threefold more than that of A. niger. These high expression levels were further confirmed by real-time PCR as well as comparison to other promoter. Moreover, the 188 bp-fragment was also examined for the promoter activity in other fungus Cochliobolus heterostrophus. Transformation of C. heterostrophus to hygromycin B resistance was successful indicating that this fragment can be applied as a strong promoter for various fungal biotechnology.