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

doi:10.7668/hbnxb.20194663

Abstract

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

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.

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Figures/Tables 13

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

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.

Fig.2 Amino acid sequence encoded by beef cattle ISG20

Fig.3 Amino acid composition of beef cattle ISG20 protein

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

Fig.5 Phosphorylation sites prediction of beef cattle ISG20 protein

Fig.6 Secondary structure prediction of beef cattle ISG20 protein

Fig.7 Tertiary structure prediction of beef cattle ISG20 protein

Fig.8 Conserved domain prediction of beef cattle ISG20 protein

Fig.9 Interaction analysis of beef cattle ISG20 protein

Fig.10 Phylogenetic tree of ISG20 proteins in different species

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.

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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