Cloning and prokaryotic expression analysis of laccase gene Aclac from Auricularia cornea

【Objective】 The cloning and prokaryotic expression analysis of the laccase gene(Aclac) of the Auricularia cornea provided a basis for in-depth study of the biological function of the laccase gene in the development of the fruit body of A.cornea.【Method】 Aclac gene of A.cornea was cloned, and the full-length cDNA sequence of the gene was obtained and analyzed by bioinformatics. The target gene was also ligated to the pET-28a plasmid to construct the prokaryotic expression vector pET28a-Aclac, and the recombinant plasmid was induced to express in Escherichia coli BL21 (DE3). 【Result】 The cloned Aclac gene cDNA sequence was 1734 bp long, encoded 577 amino acids, DNA sequence was 2521 bp, contained 14 introns, the theoretical AcLAC protein molecular weight was 64.75 kD, the isoelectric point was 5.70, the instability index was 34.01, there was no transmembrane region, and it contained a signal peptide.it was highly conserved in 4 copper ion binding regions, and contained the conservative functional domains of the Cupredoxin superfamily protein. The results of phylogenetic analysis showed that AcLAC protein and fungal laccase protein were clustered together. Among them, AcLAC protein had the closest relationship with the Auricularia. AcLAC fusion protein was successfully expressed in E. coli BL21(DE3), and its molecular weight was 67 kD.【Conclusion】 The cloned Aclac gene belongs to the Cupredoxin superfamily gene and can be expressed heterologously in a prokaryotic expression system. It is speculated that it has the same biological function as other fungal laccase genes, and enriches fungal laccase resources.