Cloning and Expression Analysis of Versatile Peroxidase from Pleurotus tuber-regium
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Abstract:
Primers were designed according to the conserved domain of versatile peroxidase gene cDNA sequences of white-rot fungi, and the gene of versatile peroxidase from Pleurotus tuber-regium (termed Pt-vp1; GenBank accession number: MN701764) was obtained via amplification using degenerate PCR, rapid amplification of cDNA end (RACE), and fusion primer and nested integrated PCR (FPNI-PCR). The full-length DNA of Pt-vp1 was 1833 bp, with 15 exons and 14 introns. The length of cDNA and open reading frame were both 1077 bp and encoded 358 amino acids. Phylogenetic analysis of the amino acid sequences of VPs from various white-rot fungi showed that Pt-vp1 protein was the closest to Pleurotus ostreatus VP (GenBank accession number: ASU87523.1). The VP activity of P. tuber-regium reached the highest to 43.83 U/L after 4 days of stationary culture in the fermentation medium. Mn2+ was added at different concentrations. When its concentration was 200 μmol/L, the VP activity was the highest 91.98 U/L. The results indicated that Mn2+ was not the necessary factor for producing VP in Pleurotus tuber-regium, but could increase significantly the enzyme production. The effect of Mn2+ at different concentrations on the transcription level of Pt-vp1 was examined using quantitative real-time PCR (qRT-PCR), and the results showed that Mn2+ promoted the expression of Pt-vp1. When the concentration of Mn2+ was 300 μmol/L, the transcription level of Pt-vp1 increased 5.79 times. This study provides important information for investigating the expression mechanism and function of the VP gene from P. tuber-regium.
