Molecular cloning and characterization of the nitric oxide synthase gene from kuruma shrimp, Marsupenaeus japonicus

Abstract

Nitric oxide (NO) signaling is involved in many physiological processes in vertebrates and invertebrates. In crustaceans, nitric oxide synthase (NOS) plays a significant role in the regulation of the nervous system and in innate immunity. Here, we describe the entire cDNA sequence (4616 bp) of the kuruma shrimp Marsupenaeus japonicus NOS (Mj NOS) generated using the reverse transcriptase-polymerase chain reaction (RT-PCR) and 5′- and 3′- rapid amplification PCRs of cDNA ends from brain and gill mRNAs. The open reading frame of Mj NOS encoded a protein of 1187 amino acids with an estimated mass of 134 kDa, and had an 82.3% sequence homology with the NOS gene of the land crab Gecarcinus lateralis. Highly conserved amino acid sequences in heme and tetrahydrobiopterin were observed in the oxygenase domain. FMN, FAD and NADPH were found in the reductase domain. Mj NOS mRNA was constitutively expressed in the brain, gill, intestine, thoracic ganglion and testis of the kuruma shrimp. When Vibrio penaeicida was injected into the kuruma shrimp, Mj NOS was expressed in the brain, gill, heart, lymphoid organ, intestine and thoracic ganglion. Mj NOS expression in the gill reached its peak 12 h and decreased to its normal level 24 h after V. penaeicida injection.