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

作　　者：李晓彤 王鹏宇 方颖妍 于鸿希 张毅 王雅春 张元沛 李彦芹[3] 姜力 LI Xiaotong;WANG Pengyu;FANG Yingyan;YU Hongxi;ZHANG Yi;WANG Yachun;ZHANG Yuanpei;LI Yanqin;JIANG Li(State Key Laboratory of Animal Biotech Breeding,College of Animal Science and Technology,China Agriculture University,Beijing 100193,China;Shandong OX Livestock Breeding Co.,Ltd.,Jinan 250100,China;Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Jinan 250100,China)

机构地区：[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

出　　处：《畜牧兽医学报》2025年第4期1981-1988,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：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)。

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。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 as-sociation 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 affect-ing the mitochondrial oxidative phosphorylation process.These findings provide valuable infor-mation for the molecular breeding of frozen semen quality traits in Holstein bulls.

关 键 词：荷斯坦牛 精子 耐冻性 功能基因 分子标记 

分 类 号：S823.3[农业科学—畜牧学]