鸡E2F1和TBP蛋白的相互作用及其基因组织表达特性分析  

Interaction Between Chicken(Gallus gallus)E2F1 and TBP Protein and Analysis of Their Gene Expression Characteristics in Tissues

作　　者：唐宏 王燕碧 赵采芹 王小亚 石海英 邢静如 段志强[1,2] TANG Hong;WANG Yan-Bi;ZHAO Cai-Qin;WANG Xiao-Ya;SHI Hai-Ying;XING Jing-Ru;DUAN Zhi-Qiang(Key Laboratory of Plateau Mountain Animal Genetics,Breeding and Reproduction,Ministry of Education/Guizhou Province Key Laboratory of Animal Genetics,Breeding and Reproduction,Guizhou University,Guiyang 550025,China;College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵阳550025 [2]贵州大学动物科学学院,贵阳550025

出　　处：《农业生物技术学报》2023年第5期1022-1031,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31960698;31760732);贵州省省级科学技术项目(黔科合基础[2020]1Y134号);贵州大学培育项目(贵大培育[2021]14号)。

摘　　要：E2F转录因子1(E2F transcription factor 1,E2F1)和TATA结合蛋白(TATA-binding protein,TBP)共同参与了基因转录调节。为探究鸡(Gallus gallus)E2F1和TBP蛋白的相互作用及其基因组织表达特性,本研究通过构建鸡E2F1和TBP基因的重组真核表达载体,共同转染人胚胎肾细胞(HEK-293T)后,利用间接免疫荧光和免疫共沉淀(co-immunoprecipitation,Co-IP)实验验证两者的相互作用;采用qPCR检测鸡E2F1、TBP基因在胚胎发育14 d(E14 d)、出壳后1日龄(H1 d)、7日龄(H7 d)和14日龄(H14 d)不同组织中的表达情况。结果表明,成功构建了鸡E2F1和TBP基因的重组真核表达载体pCMV-HA-E2F1、pCMV-Myc-TBP,其在细胞中得到正确表达且两者均主要定位在细胞核。Co-IP结果表明,鸡E2F1和TBP蛋白存在相互作用。组织表达特性分析表明,鸡E2F1和TBP基因在4个阶段的不同组织中均有表达,且其表达水平存在显著差异(P<0.05);在E14 d时,E2F1和TBP基因在肺脏中表达较高,在脑组织中的表达较低;H1 d时,两者在肝脏中表达较高,在脑组织中的表达较低;H7 d时,两者在脑组织中表达较高,在腿肌中的表达较低;H14 d时,两者在肺脏、肝脏和心脏中表达较高,在胸肌、腿肌中的表达较低。鸡E2F1和TBP蛋白在细胞核中存在相互作用,E2F1和TBP基因在4个阶段的多种组织中均有表达,但以肺脏和肝脏中的相对表达较高。本研究为E2F1和TBP蛋白互作调控鸡肺脏和肝脏组织发育的作用机理提供参考。E2F transcription factor 1(E2F1)and TATA-binding protein(TBP)jointly participate in the gene transcription regulation.To investigate the interaction between chicken(Gallus gallus)E2F1 and TBP proteins and their gene expression characteristics in tissues,in this study,the recombinant eukaryotic expression vectors of chicken E2F1 and TBP genes were constructed and then transfected into human embryonic kidney cells(HEK-293T).Indirect immunofluorescence and co-immunoprecipitation(Co-IP)assays were performed to verify their interaction.In addition,qPCR assay was used to examine the expression levels of E2F1 and TBP genes in different tissues at 14 embryonic days(E14 d)in chick embryos and 1-day-old(H1 d),7-day-old(H7 d),14-day-old(H14 d)after shelling.The results showed that the recombinant vectors pCMV-HA-E2F1 and pCMV-Myc-TBP of chicken were successfully constructed,and the recombinant proteins HA-E2F1 and Myc-TBP were correctly expressed and mainly localized in the nucleus in plasmid-transfected cells.The results of tissue characteristics analysis showed that chicken TBP and E2F1 genes were expressed in different chicken tissues at 4 stages,and the expression levels were significant different(P<0.05).At E14 d,the expression of TBP and E2F1 genes was higher in lung,but lower in brain;at H1 d,the 2 gens had high expression level in liver,but low in brain;at H7 d,they showed high expression in brain,but low in leg muscles;at H14 d,there were high expression in lung,liver and heart,but low in chest and leg muscles.Chicken E2F1 protein could interact with TBP protein in the nucleus,and the expression levels of E2F1 and TBP genes were relatively high in lung and liver at different developmental stages.The results provide references for further studying the mechanism of E2F1 and TBP protein interaction regulating the development of chicken lung and liver tissues.

关 键 词：鸡 转录因子E2F1(E2F1) TATA结合蛋白(TBP) 蛋白相互作用 亚细胞定位 

分 类 号：S831.2[农业科学—畜牧学]