Moleculer Cloning And Functional Analysis On Genes Related To NF-kB Signal Pathway Of Orange-spotted Grouper,Epinephelus Coioides

Orange-spotted grouper, Epinephelus coioides, is an important marine fish in the economy of China and Southeast Asian. In recent years, with the rapidly enhanced marine farming activities, outbreaks of various pathogenic diseases have affected the grouper aquaculture industry causing huge economic losses. Singapore grouper iridovirus (SGIV) is newly emerged viral pathogens, which leads to more than90%mortality of infected groupers.NF-κB belongs to a conservative signaling pathway, which is involved in both innate immune responses and adaptive immune responses. In this study, two types of IκBα orthologues (EcIκBαA and EcIκBαB) and an IκB kinase homologue (EcIKKa) from orange-spotted grouper Epinephelus coioides were cloned and characterized. They are important inhibitors in the NF-κB signaling pathway. Furthermore, we also detected the two types of EcIκBα function during SGIV infection. Thus, this work shed light on the understanding of the NF-κB signaling regulation in grouper immune system against virus infection.EcIκBαA and EcIκBαB encoded putative proteins containing308and318amino acids, which shared59%and53%sequence identity to IκBαA and IκBαB of Danio rerio, respectively. Amino acid sequence alignment showed that both EcIκBαA and EcIκBαB contained a conserved degradation motif DSGLDS in the N-terminal region and a PEST sequence in the C-terminal region. In addition, EcIκBαA and EcIκBαB contained5and6ankyrin repeats, respectively. The genomic organisation of EcIκBαA and EcIκBαB consist of6exons and5introns. Both genes were ubiquitous transcribed, especially in tissues with related to immunization. The expression levels of EcIκBαA and EcIκBαB will reach a peak within the first12hours in response to various stimuli including lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). and then return to the normal state Dual-luciferase reporter assay suggested that both EcIκBαA and EcIκBαB were able to inhibit Ecc-Rel and Ecp65, which induced NF-κB promoter activity in grouper spleen (GS) cells. The inhibition of p65and C-Rel by EcIκBαA and EcIκBαB arose in a dose-dependent manner. Even more, the same dose of EcIκBαA inhibits the two genes more effective than EcIκBαB. Taken those together, it showed that EcIκBαA and EcIκBαB from grouper is a natural inhibitor of NF-κB. Subcellular localization analysis showed that EcIκBαB was present predominantly in the cytoplasm, while EcIκBαA was distributed throughout both the nucleus and the cytoplasm. Furthermore, overexpression of EcIκBαA. and EcIκBαB in GS cells inhibited the viral gene transcriptions of MCP, ORF019and ORF162of SGIV. In the conclusion, these findings suggested that both EcIκBαA and EcIκBαB were involved in grouper innate immunity against virus.The EcIKKa encoded a putative protein containing735amino acids, with a predicted molecular mass of80.85kDa, and a PI of6.74. It contained a conserved kinase domain (S_TKc) and a binding domain (IKKβ3NEMO), which is necessary in IκB phosphorylation and IKK complex formation. EcIKKa showed high sequence similarity with other identified IKKa, e.g.90%similarity with Oreochromis niloticus and89%similarity with Haplochromis burtoni. Semi-quantitative RT-PCR experiment showed a ubiquitously expression of EcIKKa, especially in immune-related tissues. To investigate further possible functions of EcIKKa, I used polyI:C stimulation in groupers. It yielded no significantly increased expression of EcIKKa compared with the control, which indicated that the processes of IκB phosphorylation.In summary, the orange-spotted grouper possess several regulatory elements of the NF-κB signaling pathway. The EcIκBα, an important factor of NF-κB inhibition, were present in different types and may play different roles during viral infections. The NF-κB inhibitor protein kinase, EcIKKa, may also be involved in the NF-κB signaling pathway in the host immune response to pathogene infection. These results enriched our understanding of the content of teleost NF-κB signaling pathway and provided a new theoretical basement for analysis of pathogen diseases in grouper aquacuture.