干扰ELOVL6基因对奶牛乳腺上皮细胞脂代谢相关基因的表达及甘油三酯合成的影响

Effect of ELOVL6 knockdown on the gene expression involved in lipid metabolism and triacylglycerol accumulation in dairy cow mammary epithelial cells

为揭示超长链脂肪酸延伸酶6(ELOVL6)对奶牛乳腺上皮细胞脂代谢及甘油三酯含量的影响,试验采用PCR技术扩增得到ELOVL6基因CDS区,利用在线软件对蛋白的疏水性和跨膜区进行预测,合成并筛选靶向ELOVL6基因的有效siRNA,将有效siRNA转染乳腺上皮细胞,qRT-PCR检测干扰ELOVL6基因对奶牛乳腺上皮细胞乳脂代谢相关基因表达的影响,利用试剂盒检测细胞内甘油三酯含量。结果表明:(1)克隆得到全长为795 bp的奶牛ELOVL6基因(GenBank登录号:MW960011)的CDS区;经序列比对发现,奶牛的ELOVL6基因序列与牛(NM_001102155.1)、山羊(NM_001314257.1)、大羚羊(XM_040237469.1)的同源性分别为100%、96.73%、96.35%;ELOVL6蛋白的理论等电点为5.94,疏水最大值为2.467,最小值为-2.411,具有6个跨膜区域。(2)成功筛选到靶向ELOVL6基因的siRNA,干扰效率达到90%以上(P<0.05)。(3)将筛选出的siRNA转染至奶牛乳腺上皮细胞,通过qRT-PCR技术检测乳脂合成相关基因的表达,与对照组相比,干扰ELOVL6基因显著抑制了脂肪酸从头合成及去饱和相关基因乙酰辅酶A羧化酶A(ACACA)、固醇调节元件结合蛋白1(SREBF1)、过氧化物酶体增殖物激活受体γ(PPARG)、硬脂酰辅酶A去饱和酶1(SCD1)、脂肪酸转运相关基因脂肪酸结合蛋白(FABP3)、甘油三酯合成相关基因二酰甘油酰基转移酶(DGAT1)、磷酸甘油酰基转移酶6(AGPAT6)和甘油三酯水解相关基因激素敏感性脂肪酶(HSL)的表达量(P<0.05)。(4)干扰ELOVL6基因后,乳腺细胞内的甘油三酯含量显著降低(P<0.05)。上述表明,ELOVL6基因在奶牛乳腺上皮细胞中通过影响脂质代谢相关基因的表达及甘油三酯含量,进而对奶牛乳腺的脂代谢产生调控作用。

In order to reveal the effects of gene ultra-long chain fatty acid elongase 6(ELOVL6)on lipid metabolism and triacylglycerol content of bovine mammary epithelial cells,PCR was used to clone the CDS region of ELOVL6 gene.Online software was used to predict the hydrophobicity and transmembrane region of the protein.Effective siRNA targeting ELOVL6 gene was synthesized and screened for the following experiments.Then,the effective siRNA was transfected into mammary epithelial cells,and qRT-PCR was used to detect the effect of interfering ELOVL6 on the expression of genes related to milk fat metabolism in bovine mammary epithelial cells.The intracellular triacylglycerol content was detected by the kit.The results showed as follows:(1)The CDS region of ELOVL6 gene(GenBank No.:MW960011)was cloned with a total length of 795 bp.The similarity of ELOVL6 gene sequence with cow(NM_001102155.1),goat(NM_001314257.1)and antelope(XM_040237461.1)was 100%,96.73%and 96.35%,respectively.The theoretical isoelectric point of ELOVL6 protein was 5.94,the maximum hydrophobic value was 2.467,the minimum hydrophobic value was-2.411.The protein has 6 transmembrane regions.(2)The siRNA targeting ELOVL6 gene was successfully screened,and the interference efficiency reached more than 90%(P<0.05).(3)The siRNA was transfected into bovine mammary epithelial cells,and the expression of milk fat synthesis related genes was detected by qRT-PCR.Compared with the control group,interfering with ELOVL6 gene significantly inhibited the expression levels of fatty acid de novo synthesis and desaturation related genes(ACACA,SREBF1,PPARG,SCD1),fatty acid transport related genes(FABP3),triacylglycerol synthesis related genes(DGAT1 and AGPAT6),and triacylglycerol hydrolysis related genes(HSL)(P<0.05).(4)At the same time,the content of triacylglycerol in the cells was significantly down-regulated after the interference of ELOVL6 gene(P<0.05).These results indicated that ELOVL6 gene regulates lipid metabolism in bovine mammary epithelial cells by affecting the expression of lipid metabolism-related genes and the content of triacylglycerol.