De novo transcriptome assembly of the midgut glands of herbivorous land crabs, Chiromantes haematocheir, and identification of laccase genes involved in lignin degradation.

Abstract

Herbivorous land crabs such as Chiromantes haematocheir and C. dehaani show biomass-degrading activities. In this study, we performed RNA-seq analysis to detect biomass-degrading enzymes. A de novo transcriptome assembly in the midgut glands of molting and non-molting C. haematocheir crabs was constructed using RNA sequencing. Illumina sequencing generated 44,937,002 and 44,394,310 reads from the two midgut glands. In total, 178,710 contigs with an average length of 750bp and an N50 value of 1,235bp were assembled, of which 37,890 contigs were annotated using BLASTx search against the NCBI database. We identified 22 contigs (11 genes) belonging to the laccase family and 44 contigs (22 genes) belonging to the peroxidase family. Sixteen contigs (three genes) belonging to the GH9 cellulase family were also detected. We selected the gene accounting for the majority of expressed laccase and analyzed its properties. The 24131-laccase transcript (2465bp) had one complete open reading frame, nt 149-1987, encoding a protein of 613 amino acids with a deduced molecular mass of 67.708kDa. The enzyme was shown to belong to the multicopper oxidase family. The 24131-laccase protein was confirmed to have oxidation activity against 2,6-dimethoxyphenol by ectopic expression in Escherichia coli. Laccase activity was significantly enhanced by feeding land crabs with plant diets. These data suggest that the enzyme plays an important role in the digestion of lignin in the guts of land crabs.