Molecular Characterization Of Nitric Oxide Synthetase From Pocillopora Damicornis

Coral reef is one of the most important marine ecosystems worldwide.However,in recent years,the coral reef ecosystem is suffering from a variety of serious survival threats.The corals and zooxanthellae can form a symbiotic system changing dynamically,and the symbiotic homeostatic mechanism has been a hot issue of global research.Nitric oxide(NO)system may play an important role in the maintenance of the symbiotic homeostasis.Therefore,we studied the nitric oxide synthetase(NOS)of coral Pocillopora damicornis and their symbiotic zooxanthellae,and explored the sequence characteristics of NOS and their activity under heat stress.In the present study,RACE technique was used to clone the whole-length cDNA of the coral NOS(PdNOS)and its symbiotic zooxanthellae NOS.The cDNA of PdNOS gene was of 5637 bp,including 5'UTR(Untranslated region)222 bp,3'UTR 1086 bp and open reading frame(ORF)4329 bp.Its deduced polypeptide contained 1442 amino acid residues,with the molecular weight of 161.15.There were PDZ,Flavodoxin-1,FAD binding-1 and NAD binding domains predicted in PdNOS protein.The cDNA of zooxanthella NOS gene was of 3330 bp,of which 5'UTR 64 bp,3'UTR 146 bp,ORF 3120 bp.The deduced NOS protein consisted of 1039 amino acid residues with a molecular weight of 116.45 KDa,and contained Flavodoxin-1 and FAD binding-1 domains.Two recombinant proteins of PdNOS and zooxanthella NOS fragments were expressed in Escherichia coli.A12 and 24 h after heat stress,the enzyme activity of coral NOS increased significantly.The significant change of NO content was not detected during the whole heat stress process.It was found that the activity of NOS in corals increased significantly under heat stress,and the content of NO increased too,indicating that the response mechanism of corals NO system was initiated by heat stress.This study provides a basis for the study of the symbiotic system of coral-zooxanthella and provides a theoretical basis for the protection of coral reefs.