Molecular Cloning And The Preliminary Function Of The Nitric Oxide Synthase From Brine Shrimp--Artemia Sinica

Artemia was known by the name of brine shrimp that live in high-salt water. They have only innate immunity not like mammalian who can resistant to bacteria or virus by T-cells, B-cells, and a whole set of improved immune system. Artemia can defend the harmful substances only by humoral immunity and cellular immunity. Nitric oxide (NO) signaling is involved in many physiological activities in invertebrates, as the only gas molecular that acts second message, NO plays a significant role in the regulation of the nervous system and in innate immunity. Nitric oxide synthase(NOS) is a restriction enzyme affecting the synthesis of NO directly. There are three isoforms of NOS:neuronal NOS(nNOS), endothelial NOS(eNOS) and inducible NOS(iNOS). nNOS and eNOS are constitutively expressed and regulated by the concentration of intracellular Ca2+and bind with CaM. In contrast, iNOS can binds with CaM at a high affinity at low Ca2+levels. The invertebrate NOS gene has a high sequence homology with insect and vertebrate NOS genes, also has been proved to be involved in many physiological reactions.Here, we describe the entire cDNA sequence (1488bp) of the Artemia sinica NOS generated using the reverse transcriptase-polymerase chain reaction (RT-PCR) and5’-and3’-rapid amplification PCRs of cDNA ends from body mRNAs. The open reading frame of A. sinica encoded a protein of443amino acids with an estimated mass of50.7kDa, and had an88%sequence homology with the NOS gene of Penaeus monodon. The sequence has a NADPH-binding domain and a FAD-binding domain, which are exactly features of NOS domain.We found that AsNOS was related to different periods of embryonic development of Artemia sinica,it expressed a high level at Oh, and decline at other periods. Meanwhile,AsNOS increase when let the temperature down that display the important function of NOS. As an important element in innate immune,AsNOS show significant change after stimulating by Gram-positive bacteria and Gram-negative bacteria, which can prove that AsNOS play a key role in the immune system.