Molecular Characterization And Expression Analysis Of TRAF Genes Family In Yellow Catfish (Pelteobagrus Fulvidraco)

Yellow catfish(Pelteobagrus fulvidraco)is an important and famous freshwater fish which widely distributed in rivers and lakes in China.It has the characteristics of high economic value,hypoxia tolerance and delicious meat,and is widely cultivated in China.With the increase in culture area and density,bacterial and parasitic diseases frequently outbroke and caused serious economic losses.To improve the breeding efficiency of yellow catfish,it is necessary to optimize the breeding environment and understand the mechanism of innate immunity to provide a basis for the prevention and treatment of bacterial diseases.Tumor necrosis factor receptor-associated factor(TRAFs)is an intracellular signal transduction protein that plays an important role in the regulation of innate immunity,adaptive immunity,embryonic development,stress response and tissue homeostasis.However,there are few studies on TRAF gene family in teleosts.In this study,the open reading frame(ORF)sequences of nine TRAF genes were cloned and identified in yellow catfish,and the protein structures and evolutionary relationships of Pf＿TRAFs were analyzed.Subsequently,the tissue distributions of Pf＿TRAFs m RNAs were examined in healthy yellow catfish by q RT-PCR,and the changes in m RNA expressions of Pf＿TRAFs were examined in several tissues after infection by Edwardsiella ictaluri.Moreover,the subcellular localization of Pf＿TRAFs in HEK293 T cells was investigated by constructing the TRAFs-p EGFP-N1 eukaryotic vector.This study aims to investigate the molecular characteristics and m RNA expression distribution of TRAF gene family and their expression changes in response to bacterial invasion in yellow catfish and to provide basic information for exploring the role of Pf＿TRAFs genes against bacterial infection.The main results were as follows:1.Gene cloning and sequence analysis of Pf＿TRAF gene family in yellow catfishNine TRAF genes were obtained and identified,named Pf＿TRAF1,Pf＿TRAF2a,Pf＿TRAF2b,Pf＿TRAF3,Pf＿TRAF4a,Pf＿TRAF4b,Pf＿TRAF5,Pf＿TRAF6 and Pf＿TRAF7,respectively.The length of gene ORF was 1629 bp,1839 bp,1554 bp,1671 bp,1413 bp,1437 bp,1653 bp,1617 bp and 2025 bp,respectively.They encoded 542,612,517,556,470,478,550,538 and 674 amino acids,respectively.The predicted protein structures of Pf＿TRAFs have typically conserved domains compared to mammals.The phylogenetic relationships showed that TRAF genes are conserved during evolution.Gene structure,motifs and syntenic analyses of TRAFs genes showed that the exon-intron structure and conserved motifs of TRAF genes are diverse among seven vertebrate species,and the TRAFs gene family is relatively conserved evolutionarily.Among them,TRAF1 is more closely related to TRAF2 a and TRAF2 b,and they may have evolved from a common ancestor.TRAF7 is quite different and distantly related to other TRAFs.2.Analysis of Pf＿TRAF gene family m RNA expression in yellow catfishReal-time quantitative PCR(q RT-PCR)was used to detect the m RNA expression of nine Pf＿TRAFs genes in different tissues of healthy yellow catfish.The results showed that the nine Pf＿TRAFs genes were constitutively expressed in 12 tissues(gill,skin,fin,muscle,heart,intestine,gonads,liver,spleen,kidney,head kidney and brain).Among them,Pf＿TRAF1,Pf＿TRAF2a,Pf＿TRAF4a,Pf＿TRAF4b,Pf＿TRAF6 and Pf＿TRAF7were highly expressed in the gonad,and Pf＿TRAF2b,Pf＿TRAF3 and Pf＿TRAF5 were highly expressed in the spleen.Pf＿TRAF2b,Pf＿TRAF4a and Pf＿TRAF4b were highly expressed in the gill.In addition,Pf＿TRAF4a was also highly expressed in the brain,fin,liver,muscle,skin,intestine and kidney.The m RNA expressions of nine Pf＿TRAFs genes in head kidney,spleen,gill,brain,blood and skin tissues were detected by q RT-PCR at different times after infected with Edwardsiella ictaluri.The results showed that the m RNA expression of nine Pf＿TRAFs genes changed significantly in different tissues.Among them,the expression of Pf＿TRAF5 was significantly up-regulated in the head kidney,and the expression of Pf＿TRAF1,Pf＿TRAF3,Pf＿TRAF4a and Pf＿TRAF4b were significantly down-regulated at different time points.The expression levels of Pf＿TRAF1,Pf＿TRAF2b,Pf＿TRAF3 and Pf＿TRAF6 in spleen were significantly up-regulated at different time points,while those of Pf＿TRAF2a,Pf＿TRAF4a,Pf＿TRAF4b and Pf＿TRAF5 were significantly down-regulated at different time points.Only Pf＿TRAF1,Pf＿TRAF4a and Pf＿TRAF6were down-regulated in gill.The expression of Pf＿TRAF2a and Pf＿TRAF6 was significantly up-regulated.The expression of Pf＿TRAFs was significantly up-regulated or down-regulated in the brain,except Pf＿TRAF1.Six Pf＿TRAFs in blood and seven Pf＿TRAFs in skin were significantly up-regulated or down-regulated.These results suggest that Pf＿TRAFs gene may be involved in the immune response of yellow catfish after bacterial infection.3.Subcellular localization of Pf＿TRAFs gene family in yellow catfishThe subcellular localization of Pf＿TRAFs in HEK293 T cells was obtained by TRAFs-p EGFP-N1 eukaryotic vector construction,HEK293 T cell transfection,DAPI staining and laser confocal microscopy.The results showed that all nine Pf＿TRAF genes were found localized in the cytoplasm,,and Pf＿TRAF2a,Pf＿TRAF3 and Pf＿TRAF4a could also be localized in the nucleus.The results of this study suggested that the Pf＿TRAFs gene family plays important roles in the immune response against pathogen invasion and will provide basic information to further understand the roles of TRAF gene against bacterial infection in yellow catfish.