Shi Lijun, Wu Xin, Yang Yuze, Ma Zhu, Lv Xiaoqing, Liu Lin, Li Yanhua, Zhao Feng, Han Bo, Sun Dongxiao
Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, National Engineering Laboratory for Animal Breeding, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
J Anim Sci Biotechnol. 2021 Feb 1;12(1):24. doi: 10.1186/s40104-020-00540-4.
People are paying more attention to the healthy and balanced diet with the improvement of their living standards. Milk fatty acids (FAs) have been reported that they were related to some atherosclerosis and coronary heart diseases in human. In our previous genome-wide association study (GWAS) on milk FAs in dairy cattle, 83 genome-wide significant single nucleotide polymorphisms (SNPs) were detected. Among them, two SNPs, ARS-BFGL-NGS-109493 and BTA-56389-no-rs associated with C18index (P = 0.0459), were located in the upstream of 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3) gene. AGPAT3 is involved in glycerol-lipid, glycerol-phospholipid metabolism and phospholipase D signaling pathways. Hence, it was inferred as a candidate gene for milk FAs. The aim of this study was to further confirm the genetic effects of the AGPAT3 gene on milk FA traits in dairy cattle.
Through re-sequencing the complete coding region, and 3000 bp of 5' and 3' regulatory regions of the AGPAT3 gene, a total of 17 SNPs were identified, including four in 5' regulatory region, one in 5' untranslated region (UTR), three in introns, one in 3' UTR, and eight in 3' regulatory region. By the linkage disequilibrium (LD) analysis with Haploview4.1 software, two haplotype blocks were observed that were formed by four and 12 identified SNPs, respectively. Using SAS9.2, we performed single locus-based and haplotype-based association analysis on 24 milk FAs in 1065 Chinese Holstein cows, and discovered that all the SNPs and the haplotype blocks were significantly associated with C6:0, C8:0 and C10:0 (P < 0.0001-0.0384). Further, with Genomatix, we predicted that four SNPs in 5' regulatory region (g.146702957G > A, g.146704373A > G, g.146704618A > G and g.146704699G > A) changed the transcription factor binding sites (TFBSs) for transcription factors SMARCA3, REX1, VMYB, BRACH, NKX26, ZBED4, SP1, USF1, ARNT and FOXA1. Out of them, two SNPs were validated to impact transcriptional activity by performing luciferase assay that the alleles A of both SNPs, g.146704373A > G and g.146704618A > G, increased the transcriptional activities of AGPAT3 promoter compared with alleles G (P = 0.0004).
In conclusion, our findings first demonstrated the significant genetic associations of the AGPAT3 gene with milk FAs in dairy cattle, and two potential causal mutations were detected.
随着生活水平的提高，人们越来越关注健康均衡的饮食。据报道，牛奶脂肪酸（FAs）与人类的一些动脉粥样硬化和冠心病有关。在我们之前对奶牛牛奶脂肪酸进行的全基因组关联研究（GWAS）中，检测到83个全基因组显著的单核苷酸多态性（SNPs）。其中，与C18指数相关的两个SNP，即ARS-BFGL-NGS-109493和BTA-56389-no-rs（P = 0.0459），位于1-酰基甘油-3-磷酸O-酰基转移酶3（AGPAT3）基因的上游。AGPAT3参与甘油脂质、甘油磷脂代谢和磷脂酶D信号通路。因此，它被推断为牛奶脂肪酸的候选基因。本研究的目的是进一步证实AGPAT3基因对奶牛牛奶脂肪酸性状的遗传效应。
通过对AGPAT3基因的完整编码区以及5'和3'调控区的3000 bp进行重测序，共鉴定出17个SNP，其中5'调控区有4个，5'非翻译区（UTR）有1个，内含子有3个，3'UTR有1个，3'调控区有8个。使用Haploview4.1软件进行连锁不平衡（LD）分析，观察到两个单倍型块，分别由4个和12个鉴定出的SNP形成。使用SAS9.2，我们对1065头中国荷斯坦奶牛的24种牛奶脂肪酸进行了基于单基因座和单倍型的关联分析，发现所有SNP和单倍型块均与C6:0、C8:0和C10:0显著相关（P < 0.0001 - 0.0384）。此外，使用Genomatix，我们预测5'调控区的4个SNP（g.146702957G > A、g.146704373A > G、g.146704618A > G和g.146704699G > A）改变了转录因子SMARCA3、REX1、VMYB、BRACH、NKX26、ZBED4、SP1、USF1、ARNT和FOXA1的转录因子结合位点（TFBSs）。其中，通过荧光素酶测定验证了两个SNP影响转录活性，即SNP g.146704373A > G和g.146704618A > G的A等位基因与G等位基因相比，增加了AGPAT3启动子的转录活性（P = 0.0004）。
总之，我们的研究结果首次证明了AGPAT3基因与奶牛牛奶脂肪酸之间存在显著的遗传关联，并检测到两个潜在的因果突变。
