Cloning, Sequence Analysis and Chacterization of Laccase Gene in Auricularia polytrica

Abstract:

Using PCR and RACE technique, We obtain the cDNA and genomic DNA sequence of the lac1 gene from Auricularia polytrica. The length of genomic DNA is 2514 bp, which contains 14 exons and 13 introns based on the comparison of cDNA and genomic DNA sequence. The length of the lac1 cDNA sequence is 1972 bp, which includes a complete Open Rading Frame (ORF)of 1860 bp, from N0.33 to No.1890, encoding 619 amino acides, the molecular weight is about 68 kD, and isoelectric point is about 5.15. A signal peptide sequence exists in the N-terminal of the deduced amino acids, which also contains three multi-copper oxidase domains: KOG1263, SufI and pfam00394. We blasted the deduced amino acids with the fungal laccases protein sequences on the GenBank and observed that there is higher homologous similarity: the highest identity is 41% and positive is 58% respctively, and it also includes four conserved Cu-bind domains. The full length cDNA sequence of A. polytricha lac1 gene was ligated to the pPIC9K to construct expression vector pYH3660, the recombinant plasmid pYH3660 was transformed into Pichia pastoris, laccase activity was detected from the engineering strain which was induced by methanol with the highest expression level(123 IU/L). At same time, we get the anticipative protein using the Native SDS-PAGE. Based on the analysis of the sequence structure and expression characterization, we can conclude that the lac1 gene obtained from Auricularia polytrica is laccase gene.