公牛精子耐冻性相关基因多态性位点的挖掘与功能验证

doi:10.11843/j.issn.0366-6964.2025.04.044

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (4): 1981-1988.doi: 10.11843/j.issn.0366-6964.2025.04.044

公牛精子耐冻性相关基因多态性位点的挖掘与功能验证

李晓彤¹(), 王鹏宇¹(), 方颖妍¹(), 于鸿希¹, 张毅¹, 王雅春¹, 张元沛², 李彦芹³, 姜力¹^,*()

1. 中国农业大学动物科学技术学院畜禽生物育种全国重点实验室, 北京 100193
2. 山东奥克斯畜牧种业有限公司, 济南 250100
3. 山东省农业科学院畜牧兽医研究所, 济南 250100

收稿日期:2024-10-15 出版日期:2025-04-23 发布日期:2025-04-28
通讯作者: 姜力 E-mail:lixiaotong@cau.edu.cn;2022333020304@cau.edu.cn;ny18318118563@cau.edu.cn;lijiang@cau.edu.cn
作者简介:李晓彤(2004-), 女, 山东德州人, 本科生, 主要从事分子遗传育种研究, E-mail: lixiaotong@cau.edu.cn
王鹏宇(2004-), 男, 山西晋中人, 本科生, 主要从事动物分子遗传育种研究, E-mail: 2022333020304@cau.edu.cn
方颖妍(2003-), 女, 广东开平人, 本科生, 主要从事动物分子遗传育种研究, E-mail: ny18318118563@cau.edu.cn
第一联系人：
李晓彤、王鹏宇和方颖妍为同等贡献作者
基金资助:
国家重点研发计划项目(2022YFD1302200)；国家现代农业产业技术体系(CARS-36)

Mining and Functional Verification of Gene Polymorphisms Loci Related to Bull Sperm Freezability

LI Xiaotong¹(), WANG Pengyu¹(), FANG Yingyan¹(), YU Hongxi¹, ZHANG Yi¹, WANG Yachun¹, ZHANG Yuanpei², LI Yanqin³, JIANG Li¹^,*()

1. State Key Laboratory of Animal Biotech Breeding, College of Animal Science and Technology, China Agriculture University, Beijing 100193, China
2. Shandong OX Livestock Breeding Co., Ltd., Jinan 250100, China
3. Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China

Received:2024-10-15 Online:2025-04-23 Published:2025-04-28
Contact: JIANG Li E-mail:lixiaotong@cau.edu.cn;2022333020304@cau.edu.cn;ny18318118563@cau.edu.cn;lijiang@cau.edu.cn

摘要/Abstract

摘要：

旨在挖掘影响公牛精子耐冻性的关键基因和分子标记并应用于奶牛的分子育种。本研究基于前期组学研究结果挖掘到ATP5F1A、DNASE2、LPO等11个与公牛精子耐冻性相关的重要候选基因，针对其外显子设计引物进行PCR混池测序检测遗传变异位点。随后采用飞行时间质谱方法在130头健康的公牛资源群体中对重要位点进行基因型分析和关联分析。其中，精子高耐冻组公牛55头，鲜精平均活力为0.68，冻后平均活力为0.42；精子低耐冻组公牛75头，鲜精平均活力为0.65，冻后平均活力为0.32。结果最终检测到14个重要SNPs位点，其中ATP5F1A基因外显子上的rs110909003 SNP(C/T)位点与公牛精子的耐冻性显著相关(P=0.009)。利用Western blot检测该位点不同基因型公牛精子中ATP5F1A蛋白的表达，结果显示与TT型个体相比，CC型个体精子中ATP5F1A蛋白表达量显著上升。本研究挖掘到与公牛精子耐冻性显著相关的基因ATP5F1A，以及位于该基因内的重要分子标记rs110909003，推测其可能通过影响线粒体的氧化磷酸化过程影响精子的耐冻性。研究结果为荷斯坦种公牛冻后精液品质性状分子选育提供了重要信息。

关键词: 荷斯坦牛, 精子, 耐冻性, 功能基因, 分子标记

Abstract:

The study aimed to identify key genes and molecular markers that affect the freezability of bull sperm and apply them to molecular breeding of dairy cattle. In this study, 11 candidate genes related to bull sperm freezability were identified based on the previous omics study, such as ATP5F1A, DNASE2, and LPO. Primers were designed for the exons of these genes to detect genetic variation sites via PCR pooled sequencing. Subsequently, the Time-of-Flight mass spectrometry was used to genotype important loci in a resource population of 130 healthy bulls and association analysis was performed. Among them, there were 55 bulls in the high sperm freezability group, with an average fresh sperm motility of 0.68 and an average post-freeze motility of 0.42, and 75 bulls in the low sperm freezability group, with an average fresh sperm motility of 0.65 and an average post-freeze motility of 0.32. Finally, 14 significant SNPs were identified, among which the rs110909003 SNP (C/T) located in the exon of ATP5F1A was significantly associated with the bull sperm freezability (P=0.009). Western blot analysis was performed to detect the expression of ATP5F1A in sperm with different genotypes at the rs110909003 locus. The results showed a significant increase in ATP5F1A protein expression in sperm from bulls with the CC genotype compared to those with the TT genotype. This study identified ATP5F1A significantly associated with bull sperm freezability, as well as an important molecular marker, the rs110909003 SNP, located within this gene. This SNP may influence sperm freezability by affecting the mitochondrial oxidative phosphorylation process. These findings provide valuable information for the molecular breeding of frozen semen quality traits in Holstein bulls.

Key words: Holstein cattle, sperm, freezability, functional genes, molecular markers

中图分类号:

S823.3

李晓彤, 王鹏宇, 方颖妍, 于鸿希, 张毅, 王雅春, 张元沛, 李彦芹, 姜力. 公牛精子耐冻性相关基因多态性位点的挖掘与功能验证[J]. 畜牧兽医学报, 2025, 56(4): 1981-1988.

LI Xiaotong, WANG Pengyu, FANG Yingyan, YU Hongxi, ZHANG Yi, WANG Yachun, ZHANG Yuanpei, LI Yanqin, JIANG Li. Mining and Functional Verification of Gene Polymorphisms Loci Related to Bull Sperm Freezability[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1981-1988.

图/表 4

表 1

表 2

表 3

图 1

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增区域/bp Amplification region	退火温度/℃ Annealing temperature
ATP5F1A-E-1-F	GCCAGTTCCGCTATAACCAT	chr24：45850514~45851138	56
ATP5F1A-E-1-R	GCCATCTCAAGACACCACGAA
ATP5F1A-E-7-F	TGATGGATGGTGCTCATG	chr24：45842218~45842747	52
ATP5F1A-E-7-R	TTTCCATCGGTCCTATTT
DNASE2-E-4-F	CCCCTACAGACTGAGTGTTC	chr7：12677669~12678118	60
DNASE2-E-4-R	ATGGCCCTTGACTACCTC
DNASE2-E-2-F	TCCTTCCTGTCTTCCTCCAT	chr7：12677037~12677506	60
DNASE2-E-2-R	CAGGCAGGTCAGGAATCAG
LPO-E-5-F	AGCGGCAACCCTCTGATT	chr19：9227370~9227883	59
LPO-E-5-R	CTGACCTGACTGTCCTCCC
LPO-E-9-F	GTCAACCTTATTATCCCCATT	chr19：9234022~9234651	58
LPO-E-9-R	CTGACACCCCAAATCCAG
LPO-E-11-F	TGGGAAGGAGAATTGGAC	chr19：9242716~9243351	54
LPO-E-11-R	ACCACTGTAGGGCACTTT
CUTA-E-1-F	TGCAGATCCAGGTGTTGA	chr23：7593114~7593706	54
CUTA-E-1-R	GCATCCAAAACAGCGACA
ITGA6-E-1-F	GAACGGCAACCACAACTCAAGC	chr2：24178781~24179629	60
ITGA6-E-1-R	GGCTGAACTGGGGAAGGC
ITGA6-E-7-F	TTACTGTTGATGTTCTCCTTTG	chr2：24128249~24129073	54
ITGA6-E-7-R	AAAACAAGCAGCTCATTG
ACSL4-E-2-F	TTTGTTATGGTGATTGAA	chrX：58099374~58100231	57
ACSL4-E-2-R	TTTCTACTCTATTTGTCTG
ACSL4-E-9-F	ACCCCTCTACTTGCTCTT	chrX：58051804~58052313	54
ACSL4-E-9-R	GGTCCTCTGCTTGCTATA
ACSL4-E-17-F	ACATCGGAAACCATTTTA	chrX：58034060~58034806	59
ACSL4-E-17-R	GCACAAATATCAGTCCCT

基因名 Gene name	SNP ID SNP_ID	位置 Location	等位基因 Allele	突变类型 Mutation type
ATP5F1A	rs382264973	24:45850779	G/C	synonymous variant
	rs110909003	24:45842378	C/T	missense variant
DNASE2	rs42312321	7:12677843	T/C	missense variant
	rs380569688	7:12677265	T/G	splice donor 5th base variant
LPO	rs136605698	19:9227664	A/C	synonymous variant
	rs135860093	19:9234370	G/T	missense variant
	rs378135599	19:9242904	C/T	synonymous variant
CUTA	rs136744810	23:7593210	A/C	5′ UTR variant
ITGA6	rs451676306	2:24179226	G/T	5′ UTR variant
	rs209381216	2:24128761	T/C	missense variant
	rs133912789	2:24128525	A/C	synonymous variant
ACSL4	rs3423605874	X: 58099789	A/G
	rs384883555	X: 58052098	A/G	synonymous variant
	rs135718815	X: 58034271	A/G

SNP_ID	等位基因 Allele	最小等位基因频率 Minor allele frequency (MAF)		卡方值 χ²	P值 P value
SNP_ID	等位基因 Allele	LF	HF	卡方值 χ²	P值 P value
rs382264973	G/C	0.47(C)	0.49(G)	0.191	0.662
rs110909003	C/T	0.14(C)	0.31(C)	6.731	0.009^*
rs42312321	T/C	0.47(C)	0.40(C)	0.618	0.432
rs380569688	T/G	0.15(G)	0.13(G)	0.086	0.769
rs136605698	A/C	0.49(A)	0.47(C)	0.278	0.598
rs135860093	G/T	0.15(T)	0.18(T)	0.104	0.747
rs378135599	C/T	0.22(T)	0.23(T)	0.003	0.955
rs136744810	A/C	0.42(A)	0.33(A)	1.275	0.259
rs209381216	T/C	0.31(C)	0.30(C)	0.008	0.927
rs133912789	A/C	0.42(C)	0.36(C)	0.606	0.436
rs384883555	A/G	0.10(G)	0.15(G)	1.025	0.311
rs135718815	A/G	0.24(A)	0.28(A)	0.243	0.622

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公牛精子耐冻性相关基因多态性位点的挖掘与功能验证

Mining and Functional Verification of Gene Polymorphisms Loci Related to Bull Sperm Freezability

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