牛FOXP2基因的单等位基因表达和DNA甲基化状态分析

doi:10.11843/j.issn.0366-6964.2025.09.020

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4369-4378.doi: 10.11843/j.issn.0366-6964.2025.09.020

牛FOXP2基因的单等位基因表达和DNA甲基化状态分析

郑云畅¹(), 侯睿霖¹(), 梁晓贺¹, 杨利丹¹, 张银蛟¹, 霍浩楠¹, 陈玮娜², 张萃¹, 李世杰¹^,*()

1. 河北农业大学生命科学学院, 保定 071001
2. 河北大学中医学院, 保定 071000

收稿日期:2025-02-19 出版日期:2025-09-23 发布日期:2025-09-30
通讯作者: 李世杰 E-mail:zyc15831601852@163.com;13780586712@163.com;lishijie20005@163.com
作者简介:郑云畅(2000-)，女，河北廊坊人，硕士，主要从事动物分子遗传与表观遗传学研究，E-mail: zyc15831601852@163.com
侯睿霖(2002-)，男，河北秦皇岛人，硕士，主要从事动物分子遗传与表观遗传学研究，E-mail: 13780586712@163.com
第一联系人：
郑云畅和侯睿霖为同等贡献作者
基金资助:
河北省自然科学基金(C2023204145)；河北省农业产业技术体系创新团队(HBCT2024240206)；中央引导地方科技发展资金项目(246Z2914G)

Analysis of Monoallelic Expression and DNA Methylation Status of Bovine FOXP2 Gene

ZHENG Yunchang¹(), HOU Ruilin¹(), LIANG Xiaohe¹, YANG Lidan¹, ZHANG Yinjiao¹, HUO Haonan¹, CHEN Weina², ZHANG Cui¹, LI Shijie¹^,*()

1. College of Life Science, Hebei Agricultural University, Baoding 071001, China
2. College of Medical Science, Hebei University, Baoding 071000, China

Received:2025-02-19 Online:2025-09-23 Published:2025-09-30
Contact: LI Shijie E-mail:zyc15831601852@163.com;13780586712@163.com;lishijie20005@163.com

摘要/Abstract

摘要：

旨在研究叉头框P2(forkhead box P2，FOXP2)基因在牛组织和胎盘中的单等位基因表达和DNA甲基化状态。本研究采集了2个不同时期(出生0 d犊牛和4~5岁成年牛)的健康雌性荷斯坦奶牛的组织和自然分娩的足月胎盘，通过荧光定量PCR方法分析FOXP2基因牛6个组织(心脏、肝脏、脾脏、肺脏、肾脏和大脑)和胎盘中的表达；应用基于单核苷酸多态性(single nucleotide polymorphism，SNP)的RT-PCR产物直接测序的方法分析FOXP2基因在牛组织和胎盘中单等位基因表达状态；采用亚硫酸盐测序法对牛组织、精子和胎盘的DNA甲基化状态进行分析。结果表明，FOXP2基因在成年牛6个组织(心脏、肝脏、脾脏、肺脏、肾脏和大脑)中的表达水平均高于犊牛组织。基于FOXP2基因3'非编码区的1个SNP位点(g.53771685G＞A)，发现FOXP2基因在犊牛的6个组织中为单等位基因表达；而在成年牛中仅大脑组织中为单等位基因表达，其余的5个组织(心脏、肝脏、脾脏、肺脏和肾脏)中为双等位基因表达；在胎盘中，FOXP2基因呈现为母源等位基因表达。FOXP2基因X1剪接体的启动子区和5'端在犊牛和成年牛组织、胎盘和精子均处于低甲基化状态。以上研究结果表明，FOXP2基因在牛胎盘中为母源等位基因表达的印记基因，而其在组织中的单等位基因表达呈现生长时期和组织特异性，基因启动子和5'端处于低甲基化状态，提示DNA甲基化未参与调控牛FOXP2基因的单等位基因表达。

关键词: 叉头框P2(FOXP2)基因, 等位基因表达, DNA甲基化, 牛

Abstract:

The aim of this study was to investigate the monoallelic expressions and DNA methylation status of the forkhead box P2 (FOXP2) gene in bovine tissues and placentas. The bovine tissues of two growth periods (calves of 0 d old and adult cows of 4-5 years old) and full-term placentas naturally delivered were collected. The expression levels of the FOXP2 gene were analyzed in bovine 6 tissues (heart, liver, spleen, lung, kidney and brain) and placentas by qRT-PCR. The monoallelic expressions of the FOXP2 gene were analyzed using the method of direct sequencing of RT-PCR products based on the single nucleotide polymorphism (SNP). The analysis of DNA methylation status in bovine tissues, placentas and sperms was conducted using sulphite sequencing method. The results showed that the expression of FOXP2 gene in all 6 tissues (heart, liver, spleen, lung, kidney and brain) of adult bovine was higher than that of calf tissues. Based on the SNP site of g.53771685G > A situated in the 3' non-coding region of the FOXP2 gene, monoallelic expression of FOXP2 was observed in 6 tissues of calves. However, in adult cattle, FOXP2 showed monoallelic expression only in brain tissue and biallelic expression in other 5 tissues (heart, liver, spleen, lung and kidney). The FOXP2 gene was mathernally expressed in the placenta. Hypomethylation was showed at the promoter and the 5' end of FOXP2 X1 splice in bovine tissues, placentas and sperms. These results indicated that the FOXP2 gene is an imprinted gene and the maternal allele is expressed in bovine placenta, and the monoallelic expression in bovine tissues presents growth period- and tissue-specific. Hypomethylation is in the promoter and 5' regions of FOXP2 gene, indicating that DNA methylation is not involved in the regulating monoallelic expression of bovine FOXP2 gene.

Key words: forkhead box P2(FOXP2) gene, allelic expression, DNA methylation, bovine

中图分类号:

郑云畅, 侯睿霖, 梁晓贺, 杨利丹, 张银蛟, 霍浩楠, 陈玮娜, 张萃, 李世杰. 牛FOXP2基因的单等位基因表达和DNA甲基化状态分析[J]. 畜牧兽医学报, 2025, 56(9): 4369-4378.

ZHENG Yunchang, HOU Ruilin, LIANG Xiaohe, YANG Lidan, ZHANG Yinjiao, HUO Haonan, CHEN Weina, ZHANG Cui, LI Shijie. Analysis of Monoallelic Expression and DNA Methylation Status of Bovine FOXP2 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4369-4378.

图/表 5

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图 4

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	用途 Application	扩增片段/bp Amplicon size	退火温度/℃ Tm	PCR类型 Type of PCR
q-F	CAGTACGTCATAATCTTAGCCT	表达量的检测	316	60	cDNA
q-R	CCATTGCTGTCGATATGATCC
GA-F	AACCACGAGAAGTATAAC	内参基因	223	60	cDNA
GA-R	AAGCAGGGATGATATTC
g-F	CCAATCCTTCCCTCCTCAA	鉴定杂合子	659	52	DNA
g-R	TAATGAAACTAACACTATTTTTGG
c-F	GACAGCAATGGGAACAG	等位基因分析	619	53	cDNA
c-R	CATTTGCTTTCTACAGGATG
FO20-OF-1	GGTTGGGTTTGTATTGTGGA	甲基化分析	458	52
FO20-OR-1	TTTCACTCTATACACAAAAACAC
FO20-IF-1	GTTGGAAGGGAGGTTATAGT		372	53
FO20-IR-1	TCCTATAAATACAAACAACCAAAC
FOXP2-OF	GTGTGTGTTTGTGTATTAGT		595	52
FOXP2-OR	AAAAAAAAAAAACTACCAAAACTACC
FOXP2-IF	TTAAAGGTAGAAGTTGGTGTTGT		270	53
FOXP2-IR1	ACTACCAACCCCTCCCCTATC
FO249-OF-1	GGTGTGTTGTTTGTGGGTTT		478	56
FO249-OR-1	ACACATCTACTATTTCCACACACA
FO249-IF-1	GTAAGTTTGTTTATTTTTATTTTGG		293	53
FO249-IR-2	AAACAACCTCCCCTCTCTAA

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牛FOXP2基因的单等位基因表达和DNA甲基化状态分析

Analysis of Monoallelic Expression and DNA Methylation Status of Bovine FOXP2 Gene

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