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Cloning and Expression Analysis of Vv-lac3, A Novel Functional Laccase Gene Involved in Stipe Elongation, Based on the Genomic Sequence of Volvariella volvacea

Yuanping Lu, Guangmei Wu, Lingdan Lian, Lixian Guo, Zhiyun Yang, Wei Wang, Binzhi Chen, Baogui Xie

Fungal laccases always play important roles in various physiological and developmental processes. Here, we cloned a laccase gene, and named Vv-lac3, cDNA from the Volvariella volvacea, and expressed the cDNA in Pichia pastoris. The Vv-lac3 cDNA consisted of 1599 bp containing an open reading frame (ORF) encoding a 532 amino acids. The analysis of the deduced amino acids revealed that Vv-lac3 possessed a 19-amino acids signal peptide at the N-terminal end and a 513-amino acid mature protein. The Vv-lac3 contained four conserved copper-binding sites that which is the typical structure of fungal laccases. Phylogenetic analysis showed that Vv-lac3 had a high degree of identity with other basidiomycete laccases. Native-PAGE and SDS-PAGE analysis demonstrated that the product of Vvlac3 cDNA from P. pastoris was a functional laccase with a molecular mass of 65 kDa. The expression of the Vv-lac3 gene in V. volvacea increased during button stage to the elongation stage; it reached peaked in the elongation stage, and then decreased in the maturation stage, which suggests that this gene plays a regulatory role in stipe elongation in V. volvacea.