肉牛<i>ISG20</i>基因编码区的克隆及其在子宫内膜上皮细胞容受态形成中的表达分析

doi:10.7668/hbnxb.20194663

摘要/Abstract

摘要：

为探究干扰素刺激外切酶基因20(ISG20)在肉牛子宫内膜上皮细胞容受态形成中的表达及功能分析。采用RT-PCR结合测序技术获得肉牛ISG20基因的CDS区,利用生物信息学在线工具进行了ISG20基因及其所编码的蛋白质的序列分析,并利用qRT-PCR检测了ISG20基因及其相关基因在肉牛子宫内膜上皮细胞容受态形成中的表达量。结果显示,ISG20基因的CDS区长度为516 bp,可编码一条由176个氨基酸组成的无跨膜结构域且不稳定的碱性亲水性单体蛋白质。该蛋白质含有1个DEDD 核酸外切酶超家族结构域,与ISG15和MX2等10种蛋白质相互作用,主要在细胞质中发挥作用。此外,qRT-PCR结果表明,与对照组相比,ISG20基因及其相关的ISG15和MX2在孕酮(P4)联合干扰素-tau(IFN-τ)诱导的肉牛子宫内膜上皮细胞容受态形成中的表达量均显著上调。ISG20可能参与肉牛子宫内膜上皮细胞容受态的形成,并在早期妊娠等生物学过程中发挥作用。

关键词: 肉牛, ISG20, 子宫内膜上皮细胞, 细胞容受性, 生物信息学分析

Abstract:

In order to explore the expression and function of interferon-stimulated exonuclease gene 20(ISG20)in the formation of receptivity in beef cattle endometrial epithelial cells.The CDS region of the beef cattle ISG20 was obtained by RT-PCR combined with sequencing.The sequence analysis of the ISG20 and its encoded proteins was performed using bioinformatics online tools.The expression of the ISG20 and its related genes was detected by qRT-PCR in the formation of receptivity in beef cattle endometrial epithelial cells.The results showed that the length of CDS region of the ISG20 was 516 bp,which could encode a single protein consisting of 176 amino acids with no transmembrane domain and unstable basic hydrophilicity.The protein contained the DEDD(or DnaQ-like)exonuclease superfamily domain,interacted with 10 proteins,including ISG15 and MX2,and functioned mainly in the cytoplasm.In addition,the qRT-PCR results showed that compared with the control group,the expression of the ISG20 and its related ISG15 and MX2 was both significantly up-regulated in the formation of receptivity in beef cattle endometrial epithelial cells induced by progesterone(P4)-co-interferon-tau(IFN-τ).ISG20 may be involved in the formation of receptivity in beef cattle endometrial epithelial cells and play a role in biological processes such as early pregnancy.

Key words: Beef cattle, ISG20, Endometrial epithelial cells, Cell receptivity, Bioinformatics analysis

姚妮, 罗仍卓么, 包斌武, 杨易, 王兴平. 肉牛ISG20基因编码区的克隆及其在子宫内膜上皮细胞容受态形成中的表达分析[J]. 华北农学报, 2024, 39(2): 184-191. doi: 10.7668/hbnxb.20194663.

YAO Ni, LUORENG Zhuoma, BAO Binwu, YANG Yi, WANG Xingping. Cloning of Coding Region of Bovine ISG20 Gene and Its Expression Analysis in Receptivity Formation of Endometrial Epithelial Cell[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 184-191. doi: 10.7668/hbnxb.20194663.

http://www.hbnxb.net/CN/Y2024/V39/I2/184

图/表 13

表1 引物设计

Tab.1 Primer design

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	退火温度/℃ Annealing temperature	PCR产物长度/bp Product length	用途 Application
ISG20	F GAGATGAGTCACTGCTAAGA	50.0	670	基因克隆
	R TGTTCTGAAGAGACAAAAGG
VEGFA	F GTCTACCAGCGCAGCTTCTG	60.0	212	qRT-PCR
	R TGCTGGCTTTGGTGAGGTT
MMP9	F CCCATTAGCACGCACGAC	60.0	114	qRT-PCR
	R GCCCACATAGTCCACCTGA
IL-6	F CACTCCATTCGCTGTCT	60.0	227	qRT-PCR
	R GTGTCTCCTTGCTGCTT
LIF	F GGTCTTGGCGGCAGGAGTT	60.0	102	qRT-PCR
	R GTGGCACAGGTGGCGTTGA
HOXA10	F TTTCCTTGGCAAAGAGAAGGGCTTG	60.0	188	qRT-PCR
	R GACCTTACACAAACTGGAAGAGAC
ISG20	F TGAAATCCTTAGAGGCTGTGC	60.0	286	qRT-PCR
	R GGCAGCTTTACCTGTGACCC
ISG15	F ACTCCATGACGGTATCCGAG	60.0	203	qRT-PCR
	R ACCCTTGTCGTTCCTCAC
MX2	F CTTCAGAGACGCCTCAGTCG	60.0	232	qRT-PCR
	R TGAAGCAGCCAGGAATAGTG
GAPDH	F GGCATCGTGGAGGGACTTATG	60.0	186	qRT-PCR
	R GCCAGTGAGCTTCCCGTTGAG
RPS18	F GTGGTGTTGAGGAAAGCAGACA	60.0	79	qRT-PCR
	R TGATCACACGTTCCACCTCATC

图1 肉牛ISG20基因CDS区的克隆 A.ISG20基因CDS区扩增产物的凝胶电泳图;B.扩增产物测序结果:矩形框内为上下游引物序列,圆形框内为起始密码子和终止密码子,图2同。

Fig.1 Cloning of the CDS region of beef cattle ISG20 gene A.Gel electrophoresis image of ISG20 gene CDS amplification product;B.The results of amplification product sequencing:rectangular boxes indicate the upstream and downstream primer sequence,and the bases in the circular boxes show the start codon and stop codon,the same as Fig.2.

图2 肉牛ISG20基因编码的氨基酸序列

Fig.2 Amino acid sequence encoded by beef cattle ISG20

图3 肉牛ISG20蛋白质的氨基酸组成

Fig.3 Amino acid composition of beef cattle ISG20 protein

图4 肉牛ISG20蛋白质的亲/疏水性分析

Fig.4 Hydrophilicity/hydrophobicity analysis of beef cattle ISG20 protein

图5 肉牛ISG20蛋白质的磷酸化位点预测

Fig.5 Phosphorylation sites prediction of beef cattle ISG20 protein

图6 肉牛ISG20蛋白质的二级结构预测

Fig.6 Secondary structure prediction of beef cattle ISG20 protein

图7 肉牛ISG20蛋白质的三级结构预测

Fig.7 Tertiary structure prediction of beef cattle ISG20 protein

图8 肉牛ISG20蛋白质保守结构域预测

Fig.8 Conserved domain prediction of beef cattle ISG20 protein

图9 肉牛ISG20蛋白质的互作分析

Fig.9 Interaction analysis of beef cattle ISG20 protein

图10 不同物种ISG20蛋白质的系统进化树

Fig.10 Phylogenetic tree of ISG20 proteins in different species

图11 肉牛子宫内膜上皮细胞容受态诱导 A.体外培养的肉牛子宫内膜上皮细胞;B—F.VEGFA、LIF、HOXA10、IL-6和MMP9基因在P4及IFN-τ诱导子宫内膜上皮细胞的表达量。不同小写字母表示在P<0.05水平差异显著。图12同。

Fig.11 Induction of receptivity in endometrial epithelial cells in beef cattle A.Endometrial epithelial cells of beef cattle cultured in vitro;B—F.Expression of VEGFA,LIF,HOXA10,IL-6 and MMP9 genes in P4 and IFN-τ-induced endometrial epithelial cells,respectively.The different lowercase letters represent significant differences(P<0.05).The same as Fig.12.

图12 ISG20、ISG15和MX2基因在P4及IFN-τ诱导容受态的肉牛子宫内膜上皮细胞的表达

Fig.12 Expression of ISG20,ISG15 and MX2 in beef cattle endometrial epithelial cells of receptivity induced by P4 and IFN-τ

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肉牛ISG20基因编码区的克隆及其在子宫内膜上皮细胞容受态形成中的表达分析

Cloning of Coding Region of Bovine ISG20 Gene and Its Expression Analysis in Receptivity Formation of Endometrial Epithelial Cell

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肉牛ISG20基因编码区的克隆及其在子宫内膜上皮细胞容受态形成中的表达分析

Cloning of Coding Region of Bovine ISG20 Gene and Its Expression Analysis in Receptivity Formation of Endometrial Epithelial Cell

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