Gene Cloning,Expression Pattern And Promoter Activity Analysis Of All The Members Of NF-κβ Family From Spotted Sea Bass(lateolabrax Maculatus)

NF-κB,a protein complex that controls DNA transcription,is an important transcription factor in the innate immune system.Five family members have been identified,RelA(p65),RelB,C-Rel,NF-κB1(p105/p50)and NF-κB2(p100/p52).NF-κB is present in almost all types of animal cells,and it is involved in cellular responses to many stimuli,including stress,reactive oxygen species(ROS),tumor necrosis factor(TNF-α),interleukin(IL)and other pro-inflammatory.NF-κB is a pleiotropic transcription factor that is inhibited in the cytoplasm in the form of dimers under normal conditions,NF-κB dimer will enter the nucleus to regulate the transcription of target genes after be actived.The promoter or enhancer of many genes which especially related to inflammation have NF-κB binding site.Therefore,the NF-κB signal pathway plays an important role in the regulation of immune inflammatory response.In addition to immune inflammatory responses,NF-κB is also closely related to apoptosis,stress and cell cycle regulation.At present,many NF-κB of fish have been cloned,but there are still few further studies on them.In this study,the full-length c DNAs of five family members of the spotted sea bass,RelA,RelB,C-Rel,p105 and p100,were cloned by PCR and RACE technology,which were 3188 bp,3093 bp,3415 bp,3276 bp and 3167 bp,respectively.Their ORFs are 1899 bp,1812 bp,1920 bp,2856 bp and 2703 bp,encoding 632,603,639,951 and 900 amino acids,respectively.Multiple sequence alignment of NF-κB family found that NF-κB is very conserved from human to fish.The NF-κB family have conserved RHD and IPT domain which is related to the binding of target genes.The C-termini of RelA,RelB and C-Relare more diverse,while the C-termini of p100 and p105 still conserved with ankyrin repeats and death domains.p100 and p105 do not have transcriptional activation domains,they cannot induce the transcription of target genes.The results of the phylogenetic tree also showed that the evolution of NF-κB from fish to mammal was conserved,and each of the five genes was in a cluster respectively.Gene synteny found that the collinearity of NF-κB family members of spotted sea bass with humans,chickens,zebrafish,and large yellow croakers is relatively conservative,especially large yellow croakers,which are the same order as spotted sea bass,and the gene positions and orientations of the two species on the genome basically the same.The expression levels of NF-κB in the immune tissues of the spotted sea bass were quantitatively detected by q PCR after the stimulated by intraperitoneal injection of LPS.All of them were up-regulated in the intestine,gill,head kidney and spleen,thus LPS promoted the expression of NF-κB family genes and caused an inflammatory response.p100 was down-regulated in all four tissues,especially in head kidney.After intraperitoneal injection of Poly(I: C),NF-κB in the spotted sea bass was significantly up-regulated in the four immune tissues,especially in the intestine,which is an important organ in the mucosal immunity of fish.The significant changes after Poly(I: C)stimulation also indicated that NF-κB played an important role in mucosal immune tissue.After intraperitoneal injection of E.tarda,NF-κB was significantly up-regulated,and the response was more severe than that of LPS and Poly(I: C).That may be related to the latter two being pathogenic analogs.After stimulation with PGN,the expressions of RelA and RelB in the primary culture cell of the spotted sea bass were up-regulated,indicating that RelA and RelB also be activated by PGN.The results of the four stimulations indicated that the NF-κB of the spotted sea bass was affected by viruses,bacteria and their analogs.The subcellular localization analysis of RelA and RelB of spotted sea bass in HEK293 T cells showed that both RelA and RelB were localized in the cytoplasm.After stimulation with PGN,RelA and RelB were activated and entered the nuclei.The transcription factor binding sites and transcription start sites(TSS)of the promoters of RelA and RelB were predicted,and the transcription start sites of the two promoters were located at-238 and-195.It was found that there were transcription factor binding sites related to development,cell proliferation,differentiation,apoptosis,etc.on the promoters of RelA and RelB.These binding sites made the transcription of RelA and RelB be regulated.In addition,they included the NF-κB transcriptional binding site,indicating that the expression of NF-κB is also affected by each other.The activities of RelA and RelB promoters of spotted sea bass were studied in HEK293 T cells.The stimulation of LPS and PGN up-regulated the activities of the RelA and RelB promoters.After co-transfection of the spotted sea bass PGRP-SC2 into HEK293 T cells,stimulated with PGN,it was found that the PGRP-SC2 up-regulated the activity of RelA promoter.However,the up-regulation fold of the promoter activity of RelB was reduced.PGRP-SC2 is an intracellular pattern recognition protein with bactericidal effect,which can recognize PGN.NF-κB can be activated by the stimulation of many pattern recognition receptors.The results of this study show that PGN might regulated RelA according to PGRP-SC2 in the spotted sea bass.