Molecular Cloning and Expression Analysis of the Melatonin Receptor Gene in Grass Carp (Ctenopharyngodon idella)

Abstract:

In the present study, six isoforms of melatonin receptor 1 (Mtnr1) were cloned from grass carp (Ctenopharyngodon idella) using RACE technology and bioinformatic analysis. The distribution pattern of the six paralogs in different tissues was detected using real-time quantitative polymerase chain reaction (RT-qPCR). The full-length cDNAs of the six isoforms (Mtnr1Aa, Mtnr1Ab, Mtnr1Alike, Mtnr1Ba, Mtnr1Bb, and Mtnr1C) were 2045, 2036, 2031, 2799, 2535, and 2477 bp, with open reading frames (ORF) of 1053, 1056, 1035, 1071, 1044, and 1086 bp, encoding 350, 351, 344, 356, 347, and 361 amino acids, respectively. All of the proteins contain 7 transmembrane domains (TM), an NRY motif, a CYICHS motif, a NAXXY motif, conserved residues interacting with G-proteins in the TM3, asparagine-linked glycosylation sites, and several phosphorylation sites. The six Mtnr1 proteins are composed of 20 amino acids and are stable hydrophobic proteins. Protein sequence identity analysis showed that the six Mtnr1 isoforms share 93.1%-99.4%, 84.8%-95.2%, 82.8%-96.8%, 90.1%-97.5%, 79.7%-98.3%, and 90.6%-95.6% identities with their respective counterparts in other fishes. Phylogenetic analysis revealed that six grass carp Mtnr1 paralogs were clustered into the Cyprinidae clade, sharing a close relationship with those of Danio rerio, Cyprinus carpio, and Carassius auratus. In addition, the expression of the six Mtnr1 isoform genes was detected in the liver, heart, gill, brain, muscle, forgut, midgut, hindgut, and kidney tissues. Results showed that Mtnr1Aa and Mtnr1Ba were highly expressed in the brain and kidney, suggesting that they might play a vital role in nervous system regulation and the immune response in grass carp, respectively.