干扰FADS2基因对奶山羊乳腺上皮细胞脂肪酸组成的影响

Fatty acid desaturation 2 (FADS2), as a rate-limiting enzyme in the synthesis process of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) and docosahexaenoic acid (DHA), can dehydrogenate its substrates at the 6th and 7th positions of the fatty acid chain to form double bonds. In this study, primary dairy goat (Capra hircus) mammary epithelial cells (GMECs) were used as materials, and PCR and siRNA-mediated interference techniques were utilized to study the biological characteristics and functions of FADS2 in dairy goats. In this experiment, the CDS region of the FADS2 gene (GenBank No. MK292654) was cloned and was 1,335 bp, encoding 444 amino acids, and had a high homology with sheep (Ovis aries, XM_015103138.2) and cattle (Bos taurus, NM_001083444.1); using the siRNA-mediated interference technique, when si - FADS2 was transfected into GMECs, the mRNA level of FADS2 itself was down-regulated by 60% - 70% (P < 0.01), and at the same time the protein level decreased by 15% - 18% (P < 0.05); interfering with FADS2 significantly up-regulated the expression of genes related to the synthesis of polyunsaturated fatty acids, such as the elongase of very long chain fatty acids 2 gene (ELOVL2), ELOVL6 (P < 0.01) and the fatty acid desaturation 1 gene (FADS1) (P < 0.05); at the same time, it promoted the expression of the sterol regulatory element - binding transcription protein 1a gene (SREBP1a) (P < 0.01), genes related to de novo synthesis such as the fatty acid synthase gene (FASA) and the acetyl - CoA carboxylase gene (ACACA) and the stearoyl - CoA desaturase1 gene (SCD1) (P < 0.05); interfering with FADS2 significantly reduced the proportion of AA and DHA in cells (P < 0.05), resulting in a decrease in the content of PUFAs in cells; and it inhibited the expression of the diacylglycerol acyltransferase 1 gene (DGAT1) (P < 0.01) and DGAT2 (P < 0.05), reducing the content of triacylglyceride (TAG) in cells (P < 0.05). Therefore, FADS2 has an important function in regulating the synthesis of PUFAs and the metabolism of triglycerides in the mammary gland of dairy goats. This study provides an experimental basis for the research on the metabolism of PUFAs in goat milk.

脂肪酸去饱和酶2(fatty acid desaturation 2, FADS2)作为多不饱和脂肪酸(polyunsaturated fatty acids, PUFAs)如花生四烯酸(arachidonic acid, AA)和二十二碳六烯酸(docosahexaenoic acid, DHA)等合成过程中的限速酶,可在脂肪酸链的第6、7位将其底物脱氢形成双键。本研究以原代奶山羊(Capra hircus)乳腺上皮细胞(goats mammary epithelial cells, GMECs)为材料,利用PCR及siRNA介导的干扰技术研究奶山羊FADS2的生物学特性及功能。本实验克隆得到FADS2基因(GenBank No. MK292654)的CDS区为1 335 bp,编码444个氨基酸,与绵羊(Ovis aries, XM_015103138.2)和牛(Bos taurus, NM_001083444.1)具有高度同源性;利用siRNA介导的干扰技术,在GMECs中转染si-FADS2时,FADS2自身mRNA水平下调60%~70%(P<0.01),同时蛋白水平下降15%~18%(P<0.05);干扰FADS2,显著上调与多不饱和脂肪酸合成相关的基因超长链脂肪酸延伸酶2基因(elongase of very long chain fatty acids 2, ELOVL2)、ELOVL6(P< 0.01)和脂肪酸去饱和酶1基因(fatty acid desaturation 1, FADS1)(P<0.05)表达;同时促进转录因子固醇调节元件结合蛋白1a基因(sterol regulatory element-binding transcription protein, SREBP1a)(P<0.01)、从头合成相关基因脂肪酸合酶基因(fatty acid synthase, FASA)和乙酰辅酶A羧化酶基因(acetyl-CoA carboxylase, ACACA)及硬脂酰辅酶A去饱和酶1基因(stearoyl-CoA desaturase1, SCD1)的表达(P<0.05);干扰FADS2,显著降低细胞中AA及DHA比例(P<0.05),导致细胞中PUFAs含量降低;并且抑制二酰基甘油转移酶基因1(diacylglycerol acyltransferase 1, DGAT1)(P<0.01)及DGAT2(P<0.05)表达,降低细胞中甘油三酯(triacylglyceride, TAG)(P<0.05)含量。因此,FADS2具有调控奶山羊乳腺PUFAs合成及甘油三酯代谢的重要功能。本研究为羊奶PUFAs代谢研究提供了实验依据。