Tissue Expression Characteristics Analysis Of Chicken TRAF6 And TIFA Genes And Validation Of Their Protein Interactions

TRAF6 protein is an E3 ubiquitin ligase with a typical ring finger domain and acts as a key junction protein in cell signal transduction pathway,which can affect biological processes such as cell proliferation,differentiation and apoptosis,inflammatory response and oxidative stress by activating its downstream nuclear factor-κB(NF-κB),activator protein-1(AP-1),PI3K/Akt,JNK/ P38 and interferon regulatory factor signaling pathways.TIFA protein is a kind of protein that connects TRAF6 protein to IL-1 receptor-associated kinase 1 in the interleukin 1(IL-1)signaling pathway,which regulates the ubiquitination and oligomerization of TRAF6 protein to activate the NF-κB signaling pathway to promote cytokine production.At present,studies on TRAF6 and TIFA proteinsare mostly reported in human and mice,and their interactionshave only been reported in Xenopus laevis and human.However,due to the low homology of TRAF6 and TIFA proteinsamong chicken,human and Xenopus laevis,the interaction between chicken TRAF6 and TIFA proteinsremained unclear.In addition,there were no related reports about the tissue expression characteristics of chicken TRAF6 and TIFA genes and the non-synonymous single nucleotide polymorphisms(nsSNPs)of chicken TRAF6 gene.Therefore,in this study,the complete CDS regionsof chicken TRAF6 and TIFA genes were cloned and analyzed.Moreover,real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression characteristics of chicken TRAF6 and TIFA genes in various tissues at different stages of chicken development and immune organsat different ages.Meanwhile,Recombinant eukaryotic expression vectors of chicken TRAF6 and TIFA genes were constructed and transfected into cells to verify the interactionsof chicken TRAF6 protein wtith TIFA protein by fluorescence co-localization and co-immunoprecipitation(Co-IP)assays.Furthermore,the functional SNP of chicken TRAF6 gene exon region was predicted and analyzed by bioinformatics online software.The main results are as follows:1.The chicken TRAF6 and TIFA genes were successfully amplified and cloned.Sequence analysis showed that the CDS regionsof chicken TRAF6 and TIFA genes were 1638 bp and 564 bp in length,respectively,and encoded 545 and 187 amino acids.The molecular weights of chicken TRAF6 and TIFA proteinswere about 62 kDa and 22 kDa,with theoretical isoelectric point(p I)were 6.14 and 5.18,respectively.The results of secondary and tertiary structure analysis showed that chicken TRAF6 and TIFA proteinswere composed of irregular coil and alpha helix.The nucleic acid homology and genetic tree analysis revealed that chicken TRAF6 and TIFA genes had the highest nucleic acid homology with Meleagris gallopavo(96.4%and 92.2%),and the lowest homology with grouper and oryctolagus(40.6% and31.4%).respectively.qRT-PCR results showed that TRAF6 and TIFA genes were expressed in all tissues at different stages(E14d to 14d)of chicken development,with the highest expression level in lung,followed by liver and musculature.In the immune organsof different chicken ages(from 1 to 6 months),TRAF6 and TIFA genes were expressed in spleen,thymus and bursa of Fabricius,with the highest expression level in spleen,followed by thymus and bursa of Fabricius.2.The recombinant eukaryotic expression vectors pCMV-HA-TRAF6 and pEGFP-C1-TIFA of TRAF6 and TIFA genes were successfully constructed.The amino acid homology analysis showed that the homologies of TRAF6 and TIFA proteinsamong chicken,human and mammals were 76.4%～80.3% and 49.7%～53.3%,respectively,while those with Xenopus laevis were 69.4% and 49.7%,respectively.In additon,the RING,Zing-Finger and MATH domainsof chicken TRAF6 protein and the FHA functional domainsof TIFA protein had multiple amino acid site variationswhen compared to that of human,mammals and Xenopus laevis.Subcellular localization analysis indicated that both chicken TRAF6 and TIFA proteinslocalized in the nucleus.Further fluorescence co-localization and Co-IP experiments confirmed that chicken TRAF6 protein interacted with TIFA protein in the nucleus.3.The eight nsSNPs of chicken TRAF6 gene were retrieved from dbSNP database,and three harmful mutation sites,rs736890315(S17R),rs734934585(C20G)and rs734774178(V472E)were screened from nsSNPs loci,of which rs734774178(V472E)was a common locus screened by various software.Protein stability analysis showed that all site mutationsreduced the stability of TRAF6 protein.Conservative analysis showed that D16 G,S17R and A307 T were not conserved sites,and C20G,S40G,S40 R,T42A and V472 E were conserved functional residues.Mut Pred2 prediction results revealed that V472 E mutation site led to changes in protein stability,while other mutation sites had no significant effect.The results of secondary structure analysis found that the chicken TRAF6 protein was composed of irregular coil and αhelix.However,the mutationsat V472 E,S40G and C20G all led to the decrease of rregular coil and the increase of α helix.It was strange that the change from valine to glutamate at V472 E site did not cause the local conformation change of chicken TRAF6 protein.These results clarified the tissue expression characteristics of chicken TRAF6 and TIFA genes,and confirmed the interaction between chicken TRAF6 and TIFA proteins.This study will lay a foundation for further research on the mechanism of chicken TRAF6 and TIFA genes in chicken tissue development and immune regulation.