Shaanxi Provincial Key Laboratory of Agricultural Molecular Biology, College of Animal Science and Technology, Northwest A & F University, Yangling 712100, PR China.
Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Southwest Minzu University, Chengdu, Sichuan 610041, PR China.
J Anim Sci. 2023 Jan 3;101. doi: 10.1093/jas/skad030.
Goat milk provides benefits to human health due to its richness in bioactive components, such as polyunsaturated fatty acids (PUFAs). The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in PUFAs biosynthesis. However, its role and transcriptional regulation mechanisms in fatty acid metabolism in dairy goat remain unclear. Here, our study revealed that the FADS2 gene was highly expressed during the peak lactation compared with the dry period, early lactation, and late lactation. The content of triacylglycerol (TAG) was enhanced with the increasing mRNA expression of TAG synthesis genes (diacylglycerol acyltransferase 1/2, DGAT1/2) in FADS2-overexpressed goat mammary epithelial cells (GMECs). Overexpression of FADS2 was positively correlated with the elevated concentrations of dihomo-gamma-linolenic acid (DGLA) and docosahexaenoic acid (DHA) in GMECs. BODIPY staining showed that FADS2 promoted lipid droplet accumulation in GMECs. To clarify the transcriptional regulatory mechanisms of FADS2, 2,226 bp length of FADS2 promoter was obtained. Deletion mutation assays revealed that the core region of FADS2 promoter was located between the -375 and -26 region, which contained SRE1 (-361/-351) and SRE2 (-191/-181) cis-acting elements of transcription factor sterol regulatory element-binding protein 1 (SREBP1). Overexpression of SREBP1 enhanced relative luciferase activity of the single mutant of SRE1 or SRE2, vice versa, and failed to alter the relative luciferase activity of the joint mutant of SRE1 and SRE2. Chromatin immunoprecipitation (ChIP) and site-directed mutation assays further demonstrated that SREBP1 regulated the transcription of the FADS2 gene by binding to SRE sites in vivo and in vitro. In addition, the mRNA levels of FADS2 were significantly decreased by targeting SRE1 and SRE2 sites in the genome via the CRISPR interference (CRISPRi) system. These findings establish a direct role for FADS2 regulating TAG and fatty acid synthesis by SREBP1 transcriptional regulation in dairy goat, providing new insights into fatty acid metabolism in mammary gland of ruminants.
羊奶因其富含生物活性成分，如多不饱和脂肪酸（PUFAs），对人类健康有益。脂肪酸去饱和酶 2（FADS2）是 PUFAs 生物合成的限速酶。然而，它在乳用山羊脂肪酸代谢中的作用和转录调控机制尚不清楚。本研究显示，FADS2 基因在泌乳高峰期的表达水平显著高于干奶期、泌乳早期和泌乳晚期。在 FADS2 过表达的山羊乳腺上皮细胞（GMEC）中，TAG 合成基因（二酰基甘油酰基转移酶 1/2，DGAT1/2）的 mRNA 表达增加，导致三酰基甘油（TAG）含量升高。FADS2 的过表达与 GMEC 中二高-γ-亚麻酸（DGLA）和二十二碳六烯酸（DHA）浓度的升高呈正相关。BODIPY 染色显示 FADS2 促进 GMEC 中脂滴的积累。为了阐明 FADS2 的转录调控机制，获得了 FADS2 启动子 226bp 的长度。缺失突变试验表明，FADS2 启动子的核心区域位于-375 到-26 区域，包含转录因子固醇调节元件结合蛋白 1（SREBP1）的 SRE1（-361/-351）和 SRE2（-191/-181）顺式作用元件。SREBP1 的过表达增强了 SRE1 或 SRE2 单一突变体的相对荧光素酶活性，反之亦然，而联合突变体的相对荧光素酶活性则没有改变。染色质免疫沉淀（ChIP）和定点突变试验进一步证明，SREBP1 通过在体内和体外与 SRE 结合位点结合来调节 FADS2 基因的转录。此外，通过 CRISPR 干扰（CRISPRi）系统靶向基因组中的 SRE1 和 SRE2 位点，FADS2 的 mRNA 水平显著降低。这些发现确立了 FADS2 通过 SREBP1 转录调控在乳用山羊中调节 TAG 和脂肪酸合成的直接作用，为反刍动物乳腺脂肪酸代谢提供了新的见解。
