The use of amplified flanking region-PCR in the isolation of laccase promoter sequences from the edible fungus Pleurotus sajor-caju

Aims: To determine the regulation of laccase isozyme gene transcription in Pleurotus sajor-caju in response to different aromatic inducers and physiological parameters. Methods and Results: The promoter regions for each of four different laccase isozymes were cloned from P. sajor-caju, using amplified flanking region-PCR (AFR-PCR). Sequences stretching 724, 214, 840 and 1740 bp upstream from the predicted start codons for lac1, lac2, lac3 and lac4, respectively, were cloned in each case and analysed for the presence of putative transcriptional response elements. A number of putative response elements including metal response elements, xenobiotic response elements and antioxidant response elements appear to be present. In addition putative consensus sequences such as those for the binding of AP1, AP2, creA and NIT2 transcription factors, which are involved in nitrogen and carbon regulation in different fungi, are also present in the promoter regions of some of the isozymes. Conclusions: These elements may be involved in the transcriptional regulation of laccase gene expression in P. sajor-caju. Significance and Impact of the Study: The presence of a number of putative transcriptional response elements in the promoter regions of different isozyme genes indicates a potential role for these sites in regulating laccase gene transcription in P. sajor-caju. In addition this work demonstrates the potential usefulness of AFR-PCR as a technique to clone fungal DNA sequences located upstream from known sequences.