转录因子TEAD4对猪早期胚胎发育的调控  

Transcription Factor TEAD4 Regulates Early Embryonic Development in Pigs

作　　者：张丹丹 许腾腾 高迪 齐昕 宁伟 汝振远 张翔栋 郭腾龙 申屠璐燕 于童 马洋洋 李运生 张运海 曹祖兵 ZHANG DanDan;XU TengTeng;GAO Di;QI Xin;NING Wei;RU ZhenYuan;ZHANG XiangDong;GUO TengLong;SHENTU LuYan;YU Tong;MA YangYang;LI YunSheng;ZHANG YunHai;CAO ZuBing(Key Laboratory of Conservation and Biological Breeding of Local Livestock and Poultry Genetic Resources/College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036)

机构地区：[1]安徽农业大学动物科技学院/地方畜禽遗传资源保护与生物育种安徽省重点实验室,合肥230036

出　　处：《中国农业科学》2021年第20期4456-4465,共10页Scientia Agricultura Sinica

基　　金：安徽省科技重大专项(18030701185);安徽省留学回国人员创新创业扶持计划项目(2020LCX015);合肥市留学回国人员创新创业扶持计划项目(03082009);国家自然科学基金青年基金(3180130169和31902226)。

摘　　要：【背景】众所周知TEA结构域转录因子4(TEA domain transcription factor 4,TEAD4)是TEAD转录因子家族中的一员,在决定啮齿类动物着床前胚胎的特性中发挥关键作用。在小鼠胚胎中发现,可以通过促进Cdx2表达来参与调控着床前胚胎滋养层细胞的谱系分化。若当小鼠胚胎中缺乏TEAD4时可导致小鼠囊胚形成失败。然而,TEAD4在猪早期胚胎发育中的作用尚不清楚。【目的】通过初步阐明TEAD4对猪早期胚胎发育的影响,以期为进一步探索转录因子对猪早期胚胎发育的分子机制奠定理论基础。【方法】利用网页版工具对猪TEAD4进行生物信息学分析,主要包括对猪TEAD4序列的分析,猪与人、小鼠之间同源性的比较,以及TEAD4在不同物种之间进化关系的比较。再通过试验检测TEAD4在猪早期胚胎发育中的作用。首先采用荧光定量PCR技术检测TEAD4在猪卵母细胞和早期胚胎中mRNA表达水平,再通过设计靶向TEAD4的siRNA,采用显微注射技术注入成熟卵母细胞中,降低卵胞质内源性的TEAD4水平,并确定TEAD4 siRNA仅作用于TEAD4,以期确定TEAD4在猪早期胚胎发育中的作用。【结果】序列分析结果显示:猪TEAD4包含11个外显子,定位于5号染色体上,跨越长度37.188 kb,mRNA全长1473 bp,编码区全长1305 bp,编码434个氨基酸;与人、小鼠的同源性分析揭示TEAD4在不同物种中的保守性较高;且在猪和牛上的亲缘关系最近。荧光定量PCR检测基因表达水平结果显示:TEAD4 mRNA在猪卵母细胞和早期胚胎中均有表达,且以GV期卵母细胞为参照时比较发现,MII期卵母细胞表达量最低,并保持较低水平直至4-细胞时期,但到8-细胞时期表达量达到最高,而到桑椹胚和囊胚时期又逐渐下降。通过显微注射靶向TEAD4的siRNA发现:TEAD4 siRNA仅作用于卵母细胞中内源性的TEAD4,而对TEAD1和TEAD3不发挥作用;并与对照组和阴性对照siRNA组相比,注射TEAD4 siRNA显著降低8-细�【Background】TEA domain transcription factor 4(TEAD4)is known to be a member of the TEAD family of transcription factors and plays a key role in determining the characteristics of the preimplantation embryo in rodents.In mouse embryos,it was found to be involved in regulating the genealogical differentiation of trophectoderm cells in preimplantation embryos by promoting Cdx2 expression.The absence of the TEAD4 gene in mouse embryos can lead to failure of mouse blastocyst formation.However,the role of TEAD4 in early porcine embryonic development is still unclear.【Objective】This study aimed to preliminarily elucidate the effect of TEAD4 on early porcine embryonic development,in order to lay the theoretical foundation for further exploring the molecular mechanisms of transcription factors on early porcine embryonic development.【Method】In this study,the bioinformatics analysis of the porcine TEAD4 gene was performed by using web-based tools,including analysis of the porcine TEAD4 gene sequence,comparison of homology between pigs,human and mice,and comparison of the evolutionary relationship of TEAD4 between different species.The role of TEAD4 in early embryonic development in pigs was then tested.The mRNA expression level of TEAD4 gene in porcine oocytes and the early embryos was firstly detected by fluorescence quantitative PCR.and then,siRNA targeting TEAD4 was designed and injected into mature oocytes by microinjection technique to reduce the level of endogenous TEAD4 gene in the oocyte cytoplasm,and to determine that TEAD4 siRNA acts only on TEAD4 gene,with a view to determining the role of TEAD4 gene in early porcine embryonic development.【Result】Sequence analysis showed that the porcine TEAD4 gene contained 11 exons and localized on chromosome 5,with spanning 37.188 kb,1473 bp in full mRNA length,and 1305 bp in full coding region,which encoded 434 amino acids.Homology analysis with human and mouse revealed that TEAD4 was highly conserved in different species and had the closest affinity on pig an

关 键 词：猪 早期胚胎 TEAD4 孤雌激活 体外受精 

分 类 号：S828[农业科学—畜牧学]