摘要
【目的】分析肿瘤坏死因子受体相关因子6(TRAF6)及具有叉头相关结构域的TRAF相互作用蛋白(TIFA)基因在贵州黄鸡不同发育阶段各组织中的表达特点,为后续开展TRAF6和TIFA基因调控鸡的生长发育及二者相互作用调控鸡相关病毒复制打下基础。【方法】利用RT-PCR扩增鸡TRAF6和TIFA基因编码区(CDS)序列,通过ProtParam、SOPMA、SWISS-MODEL及MegAlign等在线软件进行生物信息学分析;同时采用实时荧光定量PCR分别检测TRAF6和TIFA基因在鸡胚发育第14 d(E14d)、鸡出壳第1 d(H1d)、第7 d(H7d)和第14 d(H14d)不同组织中的表达情况。【结果】鸡TRAF6和TIFA基因CDS序列全长分别为1638和564 bp,编码545和187个氨基酸残基,对应的编码蛋白分子量约62和22 kD,理论等电点(p I)为6.14和5.18。鸡TRAF6蛋白二级结构中无规则卷曲占47.08%、α-螺旋占38.14%、延伸链占11.86%、β-转角占2.92%,鸡TIFA蛋白二级结构中无规则卷曲占51.87%、α-螺旋占27.81%、延伸链占16.58%、β-转角占3.74%,二者均以无规则卷曲和α-螺旋为主。TRAF6和TIFA基因核苷酸序列同源比对分析均显示鸡与火鸡的相似性最高,分别为96.4%和92.2%,符合物种进化规律,也表明TRAF6和TIFA基因在禽类中具有一定的遗传保守性。TRAF6和TIFA基因在贵州黄鸡不同发育阶段的眼球、脑组织、心脏、肝脏、肺脏、肌胃、胸肌及腿肌中均有不同程度表达,且内脏组织的相对表达量普遍高于肌肉组织,以肺脏中的相对表达量最高,其次是肝脏和肌胃;从E14d发育到H14d,鸡TRAF6和TIFA基因在肺脏、肝脏和肌胃中的表达均呈先下降后上升的变化趋势,在眼球中则呈先上升后下降再上升的变化趋势,在脑组织、胸肌和腿肌的表达水平较低且趋于稳定。【结论】TRAF6和TIFA基因在鸡不同发育阶段各组织中均有表达,且以肺脏和肝脏中的表达水平相对较高,在脑组织、胸肌和腿肌中的表达相对较低,故推测TRAF6和TIFA基因相互作用对鸡的生长发育具有调控作用。
【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%),andβ-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 at96.4%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.
作者
赵采芹
王燕碧
张福平
唐宏
周磊
韩一帆
段志强
ZHAO Cai-qin;WANG Yan-bi;ZHANG Fu-ping;TANG Hong;ZHOU Lei;HAN Yi-fan;DUAN Zhi-qiang(College of Animal Sciences,Guizhou University/Key Laboratory of Animal Genetics,Breeding and Reproduction in the Plateau Mountains Region,Ministry of Education/Key Laboratory of Animal Genetics,Breeding and Reproduction in Guizhou,Guiyang 550025,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2022年第2期546-556,共11页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31760732,31960698)
贵州省科学技术基金项目(黔科合基础[2020]1Y134号)
贵州省地方家禽产业联合攻关项目(黔财农[2020]175号)。
