Identification Of Endonuclease Domain 1 In Japanese Flounder Paralichthys Olivaceus

Japanese flounder(Paralichthys olivaceus) is one of the important fishes in the coastal fisheries of East Asian countries such as Japan, China and Korea. The global aquaculture production of Japanese flounder has significantly increased in the past two decades. With the development of aquaculture, one of the severe challenges is that fish are susceptible to pathogen invading. Understanding its immune system is crucial for combating fish diseases and promoting the development of Japanese flounder aquaculture.Fish immune system is divided into the innate immune system and adaptive immune system, also known as the nonspecific immune system and specific immune system. The innate immune system is an important subsystem of overall immune system that protects the host from infection by other organisms. It recognizes and responses to invading pathogens in a generic and immediate way and is absolutely necessary for survival of multicellular organisms, but it does not provide long-lasting or protective immunity to the host. The adaptive immune system is composed of highly specialized cells, proteins and processes that provide host with long-lasting and highly specific protection. The innate immunity is divided into physical, cellular and humoral components. Humoral components include growth inhibitors, lytic enzymes, natural antibodies, cytokines, chemokines and antimicrobial peptides.Endonuclease domain-containing 1(ENDOD1) is a member of an ancient family of DNA/RNA non-specific nucleases. Nucleases of this family are found in a large number of prokaryotic and eukaryotic organisms, sharing the following characteristics: they act on both DNA and RNA, cleaving double-stranded and single-stranded nucleic acids, and require a divalent ion such as magnesium for their activity.In previous work, microarray was conducted to detect the gene expression in Japanese flounder after Edwardsiella tarda FKC injection. ENDOD1 gene from Japanese flounder(Jf_ENDOD1) was detected with a significantly increase after E. tarda FKC injection for 6hrs and 12 hrs, suggesting that Jf_ENDOD1 may be involved in fish immune response. In this study, the cDNA sequence of Jf_ENDOD1 was determined by RACE-PCR. The Jf_ENDOD1 cDNA of Japanese flounder contains an open reading frame(ORF) of 912 bp, which encodes 303 amino acid residues. The first 27 amino acid residues were predicted to be a signal peptide. The mature Jf_ENDOD1 consisted of 276 amino acids with a calculated molecular weight of 32 KDa. The amino acid sequence of Jf_ENDOD1 showed 76% identity to that predicted in large yellow croaker Larimichthys crocea. Jf_ENDOD1 expression analysis was conducted in various tissues post E. tarda FKC injection. In RT-PCR analysis, mRNA was marginally detected at 0h in all detected tissues, while those were expressed at 6h significantly in brain, kidney, spleen and intestine, and moderately expressed in gill and skin. The significant up-regulation of mRNA was not observed in muscle and liver.Jf_ENDOD1 recombinant protein which was produced in Escherichia coli showed DNase activity. Furthermore, to evaluate the DNase activities in vivo, total proteins from Japanese flounder were extracted at 12 hrs, 24 hrs and 72 hrs after E. tarda FKC injection. The extracted protein showed higher DNase activities than the protein extracted from fish without treatment. Since the mRNA levels were significantly up-regulated after the FKC treatment, this suggested that Jf_ENDOD1 might be responsible for the activities.