转录因子TEAD4对猪早期胚胎发育的调控

doi:10.3864/j.issn.0578-1752.2021.20.018

摘要/Abstract

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1 473 bp,编码区全长1 305 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-细胞和桑椹胚时期TEAD4 mRNA表达量,敲低效率达到80%。当敲低TEAD4表达时观察猪孤雌激活和体外受精胚胎的发育效率表明,与对照组和阴性对照siRNA组相比,显著降低TEAD4 siRNA敲低组从8-细胞至囊胚阶段的发育效率。【结论】TEAD4在各物种间保守性高,在猪和牛上的亲缘性最近,TEDA4可参与调控猪早期胚胎的发育。

关键词: 猪, 早期胚胎, TEAD4, 孤雌激活, 体外受精

Abstract:

【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, 1 473 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 and cow. The results of fluorescence quantitative PCR showed that TEAD4 mRNA was expressed in both porcine oocytes and early embryos; compared with GV-stage oocytes, the expression of TEAD4 mRNA was lowest in MII-stage oocytes and remained low until the 4-cell stage, but reached the highest expression in the 8-cell stage, and then gradually decreased in the morula and blastocyst stages. Microinjection of siRNA targeting TEAD4 revealed that TEAD4 siRNA only acted on the endogenous TEAD4 gene in oocytes, but not on TEAD1 and TEAD3, and compared with the control and negative control siRNA groups, the injection of TEAD4 siRNA significantly reduced TEAD4 mRNA expression at the 8-cell and morula embryo periods. When TEAD4 gene expression was knocked down, observation of the developmental efficiency of porcine orphan activation and in vitro fertilization embryos showed that the developmental efficiency of TEAD4 siRNA knockdown group from 8-cell to blastocyst stage was significantly reduced compared to the control and negative control siRNA groups. 【Conclusion】 The results of this study indicated that the TEAD4 gene was highly conserved across species, with the closest affinity on pigs and bovine, and that TEDA4 might be involved in regulating the development of early porcine embryos..

Key words: pig, early embryos, TEAD4, parthenogenetic activation, in vitro fertilization

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. 转录因子TEAD4对猪早期胚胎发育的调控[J]. 中国农业科学, 2021, 54(20): 4456-4465.

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. Transcription Factor TEAD4 Regulates Early Embryonic Development in Pigs[J]. Scientia Agricultura Sinica, 2021, 54(20): 4456-4465.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.20.018

https://www.chinaagrisci.com/CN/Y2021/V54/I20/4456

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干扰RNA名称 siRNA name	正向序列 Sense (5′-3′)	反向序列 Antisense (5′-3′)
siRNA-1	GGCUUUGGACAAGCCCAUUTT	AAUGGGCUUGUCCAAAGCCTT
siRNA-2	GCCAGAUCUACGACAAGUUTT	AACUUGUCGUAGAUCUGGCTT
siRNA-3	GCCAUUACUCCUACCGCAUTT	AUGCGGUAGGAGUAAUGGCTT

基因名称 Gene name	序列 (5′-3′) Sequence (5′-3′)
TEAD1	Forward Primer: CCGAAGTTTGCCTCGGACT Reverse primer: ATTTTGGCGGCCGGATTTTC
TEAD3	Forward Primer: CGAGTACCAGGTTGGCATCA Reverse primer: TTGTTCTGTAGGACGCTGGC
TEAD4	Forward Primer: CATTACTCCTACCGCATCC Reverse primer: CCTGTGTGTCTCTGTTGG
EF1a1	Forward Primer: ATTGTTGCTGCTGGTGTTG Reverse primer: TCATATCTCTTCTGGCTGTAGG

比较方式 Comparison method	猪TEAD4与人TEAD4比较 pTEAD4 vs hTEAD4	猪TEAD4与小鼠TEAD4比较 pTEAD4 vs mTEAD4	小鼠TEAD4与人TEAD4比较 hTEAD4 vs mTEAD4
CDS	90.96 %	77.93 %	78.54 %
AA	95.85 %	82.95 %	83.41 %