Cloning and Expression Analysis of Vvlcc3, a Novel and Functional Laccase Gene Possibly Involved in Stipe Elongation

Volvariella volvacea, usually harvested in its egg stage, is one of the most popular mushrooms in Asia. The rapid transition from the egg stage to elongation stage, during which the stipe stretches to almost full length leads to the opening of the cap and rupture of the universal veil, and is considered to be one of the main factors that negatively impacts the yield and value of V. volvacea. Stipe elongation is a common phenomenon in mushrooms; however, the mechanisms, genes and regulation involved in stipe elongation are still poorly understood. In order to study the genes related to the stipe elongation, we analyzed the transcription of laccase genes in stipe tissue of V. volvacea, as some laccases have been suggested to be involved in stipe elongation in Flammulina velutipes. Based on transcription patterns, the expression of Vvlcc3 was found to be the highest among the 11 laccase genes. Moreover, phylogenetic analysis showed that VvLCC3 has a high degree of identity with other basidiomycete laccases. Therefore, we selected and cloned a laccase gene, named Vvlcc3, a cDNA from V. volvacea, and expressed the cDNA in Pichia pastoris. The presence of the laccase signature L1-L4 on the deduced protein sequence indicates that the gene encodes a laccase. Phylogenetic analysis showed that VvLCC3 clusters with Coprinopsis cinerea laccases. The ability to catalyze ABTS (2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation proved that the product of the Vvlcc3 gene was a functional laccase. We also found that the expression of the Vvlcc3 gene in V. volvacea increased during button stage to the elongation stage; it reached its peak in the elongation stage, and then decreased in the maturation stage, which was similar to the trend in the expression of Fv-lac3 and Fv-lac5 in F. velutipes stipe tissue. The similar trend in expression level of these laccase genes of F. velutipes suggested that this gene could be involved in stipe elongation in V. volvacea.