Mycobiology.  2013 Mar;41(1):37-41.

Cloning and Characterization of a Novel Laccase Gene, fvlac7, Based on the Genomic Sequence of Flammulina velutipes

Affiliations
  • 1Graduate School of Bio & Information Technology, Hankyong National University, Ansung 456-749, Korea. kanghw2@hknu.ac.kr

Abstract

Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases found in white-rot fungi. Here, we report the cloning and analysis of the nucleotide sequence of a new laccase gene, fvlac7, based on the genomic sequence of Flammulina velutipes. A primer set was designed from the putative mRNA that was aligned to the genomic DNA of F. velutipes. A cDNA fragment approximately 1.6-kb long was then amplified by reverse transcriptase-PCR using total RNA, which was subsequently cloned and sequenced. The cDNA sequence of fvlac7 was then compared to that of the genomic DNA, and 16 introns were found in the genomic DNA sequence. The fvlac7 protein, which consists of 538 amino acids, showed only 42~51% identity with 12 different mushroom species containing two laccases of F. velutipes, suggesting the fvlac7 is a novel laccase gene. The first 25 amino acids of Fvlac7 correspond to a predicted signal sequence, four copper-binding sites, and four N-glycosylation sites. Fvlac7 cDNA was heterologously overexpressed in an Escherichia coli system with an approximate expected molecular weight of 60 kDa.

Keyword

Flammulina velutipes; Molecular characterization; Novel laccase gene fvlac7

MeSH Terms

Agaricales
Amino Acids
Base Sequence
Clone Cells
Cloning, Organism
DNA
DNA, Complementary
Escherichia coli
Flammulina
Fungi
Introns
Laccase
Molecular Weight
Oxidoreductases
Protein Sorting Signals
RNA
RNA, Messenger
Amino Acids
DNA
DNA, Complementary
Laccase
Oxidoreductases
Protein Sorting Signals
RNA
RNA, Messenger
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