BioProject

Ganoderma sp., a polyporoid fungus of the order Polyporales has a worldwide distribution. More...

Ganoderma sp., a polyporoid fungus of the order Polyporales has a worldwide distribution. It has taken an important place in various studies during last few decades. Some of its species possess medicinal properties and others have interesting decolourative properties. Different authors informed that laccase enzymes played a main role in degradation process of xenobiotic compounds by Ganoderma strains High biodiversity of Ganoderma genus has been reported in Cuba (Minter et al., 2000). In spite of this, the diversity of laccase genes of Ganoderma sp. remain unexplored. In this study a degenerate PCR-primer pair Cu 1F/Cu 2R specific for basidiomycetes ( Luis et al, 2004), were used to assess directly the diversity of laccase genes of Ganoderma strains collected from decayed wood in Havana City. The strains were cultivated on malt extract agar and the genomic DNA was isolated from mycelium using the Wizard purification kit, Promega. Partial amplification of laccase was carried out by PCR with specific primers according to the methodology of Luis et al., 2004. The purified PCR product was cloned into pCR 4 TOPO cloning vector (Invitrogen Life Technologies ) and the purified recombinant plasmids were sequenced in both directions with M13 reverse and M13 forward at Macrogen Europe, The Netherlands. Analysis of the PCR-amplified products by agarose gel electrophoresis showed two DNA fragments with the expected-size ranging from 140 to 200 pb. After sequencing fragments of different size, the analysis of nucleotide sequences through BLAST-N program showed that the partial gene of the different strains were 87 to 80 % identical to laccase genes and mRNA sequences reported from other laccase genes of Ganoderma weberianum, Ganoderma lucidum, Ganoderma sp. Trametes sp., Lenzites gibbosa, Dichomitus squalens, Punctularia strigosozonata and uncultured fungus. The multiple alignment of deduced amino acid sequence and phylogenetic tree construction using the neighbor-joining method with bootstrapping analysis performed with Clustal W and MEGA 5.0 respectively showed the presence of different laccase genes in Ganoderma strains (between 2 and 5 genes per strain). This result supports the detection of several laccase isozymes in all the strains and their decoulorative capacity against antraquinone and azo-dyes. The laccase gene fragments characterized from Ganoderma strains in this work contained the L1 region and a section of the region L2 . These regions, as well as L3 and L4, are considered fungal laccase signature sequences and include not only residues involved in copper-binding, but also residues responsible to maintain a local three-dimensional fold characterizing the active site of the enzyme. Therefore, the novel sequences of the laccase gene fragments in Ganoderma strains may indicate the presence of laccase isozymes with novel biochemical properties. Less...