Cloning and analysis tissue expression of chicken TRAF6 and TIFA genes

【Objective】The aim of this study was to analyze the expression characteristics of the tumor necrosis factor (TNF),receptor-associated factor 6(TRAF6) and TRAF6 interactions with a forkhead associated domain protein(TIFA) genes in tissues of Guizhou yellow chickens at different developmental stages,which provided a foundation for further studying the role of TRAF6 and TIFA genes in chicken growth and development and the replication of chicken associated virus.【Method】The coding DNA sequences(CDS) of chicken TRAF6 and TIFA genes were amplified by RT-PCR and then analyzed by bioinformatics software including ProtParam,SOPMA,SWISS-MODEL and MegAlign. Quantitative real-time PCR was used to detect the expression of TRAF6 and TIFA genes in different tissues in 14 day old(E14d) chicken embryos,and at 1(H1d),7(H7d) and 14(H14d) days after hatching.【Result】The CDS regions of chicken TRAF6 and TIFA genes were 1638 and 564 bp in length,respectively,which encoded 545 and 187 amino acids. The corresponding proteins had molecular weights of about 62 and 22 kD,with theoretical isoelectric points of 6.14 and 5.18. The results of protein secondary structure analysis showed that chicken TRAF6 and TIFA proteins mainly contained irregular coil(47.08% and 51.87%),alpha helix(38.14% and 27.81%),extended chain(11.86% and 16.58%),and β -rotation (2.92% and 3.74%),respectively. Therefore,the two proteins were primarily composed of irregular coil and alpha helix. The nucleotide homology analysis of TRAF6 and TIFA genes revealed that chicken and turkey had the highest similarity at 96.4% and 92.2%,respectively,indicating a certain degree of genetic conservatism among avian species. The tissue expression analysis showed that TRAF6 and TIFA genes had different expression levels in the eyeball,brain tissue,heart, liver,lung,masticatory stomach,breast muscle and leg muscle of Guizhou yellow chickens at different developmental stages, but the relative expression level in visceral tissues was much higher than that in muscle tissues, of which the highest visceral expression level was in the lung,followed by the liver and masticatory stomach. From E14d to H14d,the expression of chicken TRAF6 and TIFA genes in lung,liver and masticatory stomach first showed a trend of decrease then an increase. The expression in the eye showed first a rising trend then falling and rising again. The expression in brain tissue, breast muscle and leg muscle was initially low then tended towardsa higher,stable level.【Conclusion】TRAF6 and TIFA genes were expressed in all tissues of chicken at different developmental stages,with relatively high expression levels in the lung and liver,but relatively low in brain tissue,breast muscle and leg muscle,suggesting that the interaction between TRAF6 and TIFA genes could regulate the growth and development of chickens.