Molecular cloning, inducible expression and function analysis of Epinephelus coioides Sec6 response to SGIV infection.

Exocyst complex component 3 Sec6 of mammals, one of the components of the exocyst complex, participates in numerous cellular functions, such as promoting cell migration and inhibiting apoptosis. In this study, the Sec6 was obtained from Epinephelus coioides, an economically important cultured fish. The full length of E. coioides Sec6 was 2655 bp including a 245 bp 5' UTR, a 154 bp 3' UTR, and a 2256 bp open reading frame (ORF) encoding 751 amino acids, with a molecular mass of 86.76 kDa and a theoretical pI of 5.57. Sec6 mRNA was detected in all the tissues examined, but the expression level is different in these tissues. Using fluorescence microscopy, Sec6 were distributed in both the nucleus and the cytoplasm. After SGIV infection, the expression of E. coioides Sec6 was significantly up-regulated in both trunk kidney and spleen response to Singapore grouper iridovirus (SGIV), an important pathogens of E. coioides. Sec6 could increase the SGIV-induced cytopathic effects (CPE), the expression of the SGIV genes VP19, LITAF, MCP, ICP18 and MCP, and the viral titers. Besides, E. coioides Sec6 significantly downregulated the promoter of NF-κB and AP-1, and inhibited the SGIV-induced apoptosis. The results demonstrated that E. coioides Sec6 might play important roles in SGIV infection.

A novel MKK gene (EcMKK6) in Epinephelus coioides: Identification, characterization and its response to Vibrio alginolyticus and SGIV infection.

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.