Wang Xinyue, Bao Jingjing, Bi Yazhen, Hu Wenping, Zhang Li
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
College of Animal Science and Technology, Qingdao Agriculture University, Qingdao 266109, China.
Biology (Basel). 2022 Dec 10;11(12):1795. doi: 10.3390/biology11121795.
Growth traits are influential factors that significantly affects the development of the sheep industry. A previous TMT proteomic analysis found that a key protein in the HIF signaling pathway, ARNT, may influence embryonic skeletal muscle growth and development in sheep. The purpose of this study was to better understand the association between the polymorphisms of ARNT and growth traits of sheep, and the potential function of ARNT. Real-time qPCR (qRT-PCR) of ARNT was carried out to compare its expression in different developmental stages of the muscle tissues and primary myoblasts in the Hu, Chinese merino, and Gangba sheep. The genetic variance of ARNT was detected using the Illumina Ovine SNP 50 K and 600 K BeadChip in the Hu and Ujimqin sheep populations, respectively. The CDS sequence of the ARNT gene was cloned in the Hu sheep using PCR technology. Finally, bioinformatic analytical methods were applied to characterize the genes and their hypothetical protein products. The qRT-PCR results showed that the ARNT gene was expressed significantly in the Chinese merino embryo after 85 gestation days (D85) (p < 0.05). Additionally, after the sheep were born, the expression of ARNT was significant at the weaning stage of the Hu sheep (p < 0.01). However, there was no difference in the Gangba sheep.In addition, six SNP loci were screened using 50 K and 600 K BeadChip. We found a significant association between rs413597480 A > G and the Hu sheep weight at weaning and backfat thickness in the 5-month-old sheep (p < 0.05), and four SNP loci (rs162298018 G > C, rs159644025 G > A, rs421351865 G > A, and rs401758103 A > G) were also associated with growth traits in the Ujimqin sheep (p < 0.05). Interestingly, we found that a G > C mutation at 1948 bp in the cloned ARNT CDS sequence of the Hu sheep was the same locus mutation as rs162298018 G > C identified using the 600 K BeadChip, which resulted in a nonconservative missense point mutation, leading to a change from proline to alanine and altering the number of DNA, protein-binding sites, and the α-helix of the ARNT protein. There was a strong linkage disequilibrium between rs162298018 G > C and rs159644025 G > A, and the ARNT protein was conserved among the goat, Hu sheep, and Texel sheep. And, we propose that a putative molecular marker for growth and development in sheep may be the G > C mutation at 1948 bp in the CDS region of the ARNT gene. Our study systematically analyzed the expression, structure, and function of the ARNT gene and its encoded proteins in sheep. This provides a basis for future studies of the regulatory mechanisms of the ARNT gene.
生长性状是显著影响绵羊产业发展的重要因素。先前的TMT蛋白质组学分析发现，低氧诱导因子（HIF）信号通路中的关键蛋白芳香烃受体核转运蛋白（ARNT）可能影响绵羊胚胎骨骼肌的生长发育。本研究旨在更好地了解ARNT基因多态性与绵羊生长性状之间的关联以及ARNT的潜在功能。通过实时定量聚合酶链反应（qRT-PCR）检测ARNT在湖羊、中国美利奴羊和岗巴羊肌肉组织及原代成肌细胞不同发育阶段的表达情况。分别利用Illumina绵羊SNP 50K和600K芯片检测湖羊和乌珠穆沁羊群体中ARNT基因的遗传变异。采用聚合酶链反应（PCR）技术克隆湖羊ARNT基因的编码序列（CDS）。最后，运用生物信息学分析方法对该基因及其假定的蛋白质产物进行特征分析。qRT-PCR结果显示，ARNT基因在中国美利奴羊妊娠85天（D85）后的胚胎中表达显著（p<0.05）。此外，绵羊出生后，ARNT在湖羊断奶阶段表达显著（p<0.01）。然而，岗巴羊中无差异。另外，利用50K和600K芯片筛选出6个单核苷酸多态性（SNP）位点。我们发现rs413597480 A>G与湖羊断奶体重及5月龄羊的背膘厚显著相关（p<0.05），4个SNP位点（rs162298018 G>C、rs159644025 G>A、rs421351865 G>A和rs401758103 A>G）也与乌珠穆沁羊的生长性状相关（p<0.05）。有趣的是，我们发现湖羊克隆的ARNT CDS序列中1948bp处的G>C突变与利用600K芯片鉴定的rs162298018 G>C为同一基因座突变，该突变导致非保守错义点突变，使脯氨酸变为丙氨酸，改变了ARNT蛋白的DNA结合位点数量、蛋白质结合位点数量及α螺旋结构。rs162298018 G>C与rs159644025 G>A之间存在强连锁不平衡，且ARNT蛋白在山羊、湖羊和特克塞尔羊中保守。并且，我们提出ARNT基因CDS区1948bp处的G>C突变可能是绵羊生长发育的一个假定分子标记。本研究系统分析了绵羊ARNT基因及其编码蛋白的表达、结构和功能，为今后研究ARNT基因的调控机制提供了依据。
