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Molecular Characterization of Laccase Genes From the Basidiomycete Trametes hirsuta Bm-2 and Analysis of the 5' Untranslated Region (5'UTR)

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Molecular Characterization of Laccase Genes From the Basidiomycete Trametes hirsuta Bm-2 and Analysis of the 5' Untranslated Region (5'UTR)

Alejandrina Pereira-Patrón et al. 3 Biotech.

Abstract

The aim of this study was to identify and characterize laccase genes produced by Trametes hirsuta Bm-2 in a liquid medium, both with and without induction. The amplification of 5'and 3'regions of laccase sequences was obtained by the RACE-PCR method, and these were assembled to obtain a cDNA of total length. Two new laccase genes were isolated from basal medium (lac-B) and lignocellulosic grapefruit substrate (lac-T), both encoding open reading frames of 2566 bp. Both laccase-predicted proteins consisted of 521 amino acids, four copper-binding regions, a signal peptide, and five potential glycosilation sites (Asn-Xaa-Ser/Tre). Moreover, the deduced amino acid sequences share about 76-85% identity with other laccases of WRF. Sequence comparison showed 47 synonymous point mutations between lac-B and lac-T. In addition, 5' untranslated regions (UTR) of laccase genes lac-B and lac-T showed differences in length and number of regulatory elements that may affect transcriptional or translational expression of these genes.

Keywords: Laccase genes; RACE-PCR; Trametes hirsuta; Untranslated regions.

Conflict of interest statement

Compliance with ethical standardsThe authors declare that there is no conflict of interest regarding publication of this paper.

Figures

Fig. 1
Fig. 1
Nucleotide and amino acid sequence of the lac-B gene. The secretion peptide is double underlined, the potential sites of N-glycosylation are shown in boxes, and the four-conserved laccase domains are underlined. An asterisk shows the stop codon. The nucleotides are differentiated with respect to the sequence of the lac-T gene
Fig. 2
Fig. 2
Nucleotide and amino acid sequence of the lac-T gene. The secretion peptide is double underlined, the potential sites of N-glycosylation are shown in boxes, and the four-conserved laccase domains are underlined. An asterisk indicates the stop codon. Highlights show the different nucleotides with respect to the sequence of the lac-B gene
Fig. 3
Fig. 3
Alignment of the amino acid sequence of laccase genes of T. hirsuta Bm-2 and other ligninolytic fungi. The identical residuals are shown with asterisks. The four-conserved laccase domains are indicated in boxes
Fig.  4
Fig.  4
Phylogenetic relationship of laccase genes of Trametes hirsuta Bm-2 with other laccase genes. The MEGA 7.0 program and the Likelihood method with 1000 bootstrap were used to construct the tree from cDNA sequences obtained from the database within the NCBI. The lac-B and lac-T genes of T. hirsuta Bm-2 are shown with circle symbols
Fig. 5
Fig. 5
Predicted secondary structure of 5′UTR regions in lac-B
Fig. 6
Fig. 6
Predicted secondary structure of 5′UTR regions in lac-T

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