Characterization of a novel ADP-ribosylation factor gene from Macrobrachium nipponense and its response to ammonia nitrogen stress

Abstract

ADP-ribosylation factors (Arfs) are guanosine triphosphate (GTP)-binding proteins that play essential roles in membrane trafficking, and they have been recently reported to be involved in innate immunity in crustaceans. However, little information is available on Arfs in the oriental river prawn Macrobrachium nipponense and their response to ammonia nitrogen stress. In this study, we identified a novel M. nipponense Arfn gene (MnArfn). The full-length cDNA of MnArfn was 1076 bp. It contained a 537 bp open reading frame (ORF) and encoded a 178 amino acid protein with a predicted molecular weight of 19.85 kDa. Sequence and phylogenetic analyses showed that MnArfn was an unidentified Arf, sharing 55-61% identity with other known Arfs. Quantitative real-time PCR (qPCR) indicated that all examined tissues (hepatopancreas, stomach, gill, heart, muscle, and eyestalk) expressed MnArfn. Hepatopancreas and gills, 2 organs involved in environmental stress management, had the highest expression. Under conditions of ammonia nitrogen stress, MnArfn expression in hepatopancreas and gills was significantly up-regulated at 6, 12, and 24 h. Western blotting experiments also revealed that MnArfn was distributed in all examined tissues, with the highest expression in hepatopancreas and gills, consistent with qPCR results. The findings from this study indicate that MnArfn may play an important role in the response of M. nipponense to ammonia nitrogen stress, which provides a new avenue to study the resistance mechanism(s) of crustaceans to ammonia nitrogen and to screen for individuals with resistance to unfavorable environments.