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

Abstract

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.

Low carbon/nitrogen ratio increases laccase production from basidiomycetes in solid substrate cultivation

“…Some of them are constitutively expressed and others are induced by physiological and growth conditions (Mansur et al, 1998, Soden andDobson, 2003;Xiao et al, 2006). The amount of N in the culture medium determines the laccase gene expression for Trametes versicolor (Collins and Dobson, 1997) and laccase production for Pycnoporus cinnabarinus (Eggert et al, 1996).…”

Identification of a new member of the dye-decolorizing peroxidase family from Pleurotus ostreatus

“…TATA (position 280 of AM283034 deposited sequence) and CAAT boxes (position 64) were identified in the 5¢ gene-flanking region, extending 400 bp upstream of the ATG. Stretches that closely match consensus sequences of regulatory elements, such as stress responsive element (STRE, positions 367 and 413) and catabolic responsive element (Cre-A, position 141) were also recognized in that region (Soden and Dobson 2003).…”

Cloning and Functional Characterization of the styE Gene, Involved in Styrene Transport in Pseudomonas putida CA-3

“…A 1.5-kb region downstream from the styD gene was cloned using amplified flanking-region PCR. The method used was a modification of that previously described (29) and included the following steps. (i) An initial round of PCR using a 5Ј biotinylated gene specific primer, StyD-BIO, and a degenerate flanking primer, primer A (Table 1), was carried out.…”