SREBP-1基因对山羊乳腺上皮细胞脂肪酸代谢的调控作用研究

发布时间：2018-05-01 19:30

本文选题：奶山羊 + SREBP1　；参考：《西北农林科技大学》2016年博士论文

【摘要】：固醇调节元件结合蛋白-1(Sterol regulatory element binding protein-1,SREBP-1)是哺乳动物体内重要的核转录因子之一,主要参与调控内源的胆固醇、脂肪酸、磷脂及甘油三酯等的生物合成及代谢。在反刍动物乳腺组织中,SREBP-1通过激活乳脂合成及分泌过程中关键基因及酶的表达,促进脂肪酸的从头合成过程,调控乳中有益脂肪酸的成分及含量,是乳脂合成的关键调控因子。羊奶含有丰富的短中链脂肪酸(C≤16),因此,通过奶山羊SREBP-1基因的功能研究解析乳腺上皮细胞脂肪酸代谢调控作用,对奶山羊育种及羊奶成分调控具有重要理论意义和实际应用价值。本研究通过腺病毒介导的超表达技术及si RNA介导的干扰技术对SREBP-1基因的基本功能进行探讨,并使用LXR-SREBP1通路的激动剂(T0901317)进行功能验证;通过对山羊SREBP-1c及ACSS2基因启动子的克隆及突变研究,探讨山羊SREBP-1c基因及ACSS2基因的转录调控机制;通过对脂肪酸从头合成过程中关键酶ACSS2及ACLY基因的干扰研究,验证其对脂肪酸从头合成及甘油三酯合成的影响。本研究的主要结果如下:1.山羊SREBP-1基因的过表达研究将克隆得到的山羊SREBP-1基因(1-1 209 bp,n SREBP1)构建到p Adtrack-CMV穿梭载体中,与骨架载体p Ad Easy-1重组后,转染HEK293细胞,包装扩增出高滴度的重组腺病毒Ad-n SREBP1,感染山羊原代乳腺上皮细胞48 h后,SREBP-1基因m RNA水平及蛋白水平显著上调,同时导致脂肪酸合成代谢相关基因:ACSL1,FABP3,ELOVL6,SCD1,ACSS2,ACLY,ACACA,FASN,IDH1,INSIG1,NR1H3,PPARG等基因的表达量显著上调(P0.05),而SREBP-1蛋白成熟裂解过程的护送蛋白SCAP的表达量则显著下调(P0.05)。同时,过表达SREBP-1基因显著上调甘油三酯合成相关基因LPIN1、DGAT1的表达量及细胞内甘油三酯的含量(P0.01),细胞内C16:0及C18:1的含量显著增加(P0.05),C16:1,C18:0及C18:2的含量显著减少(P0.05),而细胞内长链多不饱和脂肪酸(如C20:4及C22:6)的含量无明显变化(P0.05)。2.山羊SREBP-1基因的RNA干扰设计并合成特异靶向SREBP-1基因氨基末端活性区域的si RNA,稀释后转染山羊原代乳腺上皮细胞,48 h后,SREBF1及SREBP-1a基因表达量显著下调60%以上(P0.01),SREBP1蛋白成熟形式表达量显著减少。SREBP1基因的干扰导致细胞内脂肪酸合成、转运相关基因及脂代谢调控因子(SCD1,ELOVL6,ACLY,ACSS2,IDH1,FABP3,ACSL1,SLC27A6,INSIG1,SCAP,PPARG及CPT1A)的表达量显著下调(P0.05),PLIN3,XDH,PPARA及ACOX的表达量显著上调(P0.05),ACACA,FASN,CD36,GPAM,DGAT1,PLIN2,NR1H3,ATGL,HSL等基因的表达量无显著变化(P0.05)。SREBP1基因的干扰导致细胞内甘油三酯的含量显著下调(P0.05),细胞内胆固醇的含量下调。3.LXRα-SREBP1通路激活对山羊乳腺脂肪酸代谢的影响使用不同浓度LXRα特异性激动剂T0901317处理山羊乳腺上皮细胞48 h后,SREBP1基因转录水平及蛋白水平的表达量均呈梯度上升趋势,并且细胞核内成熟形式的SREBP-1蛋白积累增加。同时,T0901317的添加激活细胞内长链脂肪酸激活、转运、去饱和,脂肪酸从头合成,及甘油三酯合成等生理过程,导致SCL27A6,ACSL1,FABP3,SCD1,ELOVL6,ACSS2,ACLY,ACACA,FASN,IDH1,GPAM,LPIN1,DGAT1,DGAT2,PLIN2,PLIN3,ATGL,XDH等基因的表达量显著上调(P0.05),同时脂代谢调控因子INSIG1,SCAP,PPARA及PPARG的表达量也显著上调(P0.05)。LXRα-SREBP1通路的激活导致细胞内脂滴数量及甘油三酯含量均显著增加,细胞内C16:0及C18:2含量降低,C18:0的含量显著减少(P0.05),单不饱和脂肪酸(C16:1,C18:1,C20:1及C22:1)的含量显著增加(P0.05),长链多不饱和脂肪酸的含量显著降低(P0.01)。4.山羊SREBP-1c基因的转录调控作用克隆得到山羊SREBP-1c基因5'侧翼序列2 186 bp,含转录起始位点上游2 012 bp。生物信息学分析发现,SREBP-1c启动子序列含有两个SREBP1结合位点(SRE),两个LXR结合位点(LXRE),并且在两个SRE位点附近含有核因子Y结合位点(NF-Y)及Sp1位点。荧光素酶活性分析表明,LXR的激动剂T0901317可以显著上调SREBP-1c的启动子活性(P0.05),而SREBP1基因的si RNA导致SREBP-1c启动子活性显著下调(P0.01)。缺失突变结果表明,SREBP-1c基因启动子核心区域位于-395 bp~+1 bp;并且,当SREBP-1c启动子由-395 bp缺失到-86 bp时,T0901317对SREBP-1c基因启动子的激活作用消失。定点突变SRE及LXRE位点后转染山羊原代乳腺上皮细胞,结果表明:两个SRE位点的单突变或者双突变均导致SREBP-1c启动子基础活性显著下调(P0.01),而LXRE位点的突变对启动子活性无明显下调作用(P0.05)。使用T0901317处理后,单一位点的LXRE或者SRE位点的突变均导致T0901317对SREBP-1c启动子的激活作用减弱,而两个LXRE位点的同时突变则导致T0901317对SREBP-1c启动子的激活作用消失。结果表明,LXRα及n SREBP1均对山羊SREBP-1c基因的表达有重要的调控作用。5.山羊ACSS2基因的转录调控作用扩增得到山羊ACSS2基因启动子5'侧翼序列2 359 bp,含转录起始位点上游1 976 bp。启动子序列分析结果表明,ACSS2启动子上存在SREBP-1,LXR,NF-Y,Sp1和STAT5等转录因子结合位点。启动子荧光素酶活性分析发现,SREBP1基因的si RNA显著下调ACSS2基因启动子活性(P0.05),而LXR的特异性激动剂T0901317导致ACSS2基因启动子活性显著升高(P0.05)。缺失突变发现,ACSS2基因启动子核心区域位于-515 bp~+1 bp,并且在该区域内存在一个保守的SRE位点。对SRE位点进行定点突变后,导致ACSS2启动子活性显著下调(P0.05)。染色质免疫共沉淀实验结果表明,SREBP1蛋白与ACSS2启动子上的SRE元件序列结合,直接调控ACSS2基因的表达。6.ACSS2及ACLY共同干扰对脂肪酸及甘油三酯合成的影响设计并合成特异靶向山羊ACSS2及ACLY基因的si RNA,转染山羊原代乳腺上皮细胞48 h后,ACSS2及ACLY基因m RNA表达量显著下调70%左右(P0.05),同时,细胞内脂代谢相关基因ACACA,FASN,SCD1,ELOVL6,DGAT1,DGAT2,AGPAT6,FABP3,CD36,PPARA,ACOX,CPT1A,PLIN2,PLIN3等基因的表达量显著下调(P0.05),细胞内脂滴积累减少,甘油三酯含量降低。综上所述,SREBP1基因广泛参与调控脂肪酸生物合成、转运及甘油三酯合成等生物过程中相关基因的表达,增加细胞内单不饱和脂肪酸的含量,降低长链多不饱和脂肪酸的含量。LXRα基因可直接激活SREBP-1基因的表达,共同调控山羊乳腺脂肪酸代谢,并且山羊SREBP-1c基因同时受到LXRα及其成熟形式n SREBP1的调控。SREBP-1基因可在转录水平直接调控ACSS2基因的表达,影响脂肪酸及甘油三酯的合成。
[Abstract]:Sterol regulatory element binding protein -1 (Sterol regulatory element binding protein-1, SREBP-1) is one of the important nuclear transcription factors in mammals. It is mainly involved in the biosynthesis and metabolism of endogenous cholesterol, fatty acids, phospholipids and triglycerides. In ruminant mammary tissues, SREBP-1 is synthesized by activating milk fat. The expression of key genes and enzymes in the secretory process promotes the initio synthesis of fatty acids and regulates the composition and content of beneficial fatty acids in milk. It is a key regulator of milk fat synthesis. Sheep milk contains rich short medium chain fatty acids (C < 16). Therefore, the fatty acid generation of mammary epithelial cells is analyzed by the function of SREBP-1 gene of milk goats. It has important theoretical significance and practical application value for dairy goat breeding and the regulation of goat milk components. This study explored the basic functions of SREBP-1 gene by adenovirus mediated overexpression and Si RNA mediated interference technology, and used LXR-SREBP1 pathway agonist (T0901317) to perform functional verification. The cloning and mutation of the promoter of SREBP-1c and ACSS2 genes in goats was studied to investigate the transcriptional regulation mechanism of SREBP-1c and ACSS2 genes in goats. The effects of the ACSS2 and ACLY genes on the key enzymes of fatty acids in the initio synthesis of fatty acids were investigated to verify the effect of the gene on the synthesis of fatty acids from ab initio and glycerol three ester. The main results of this study were as follows Under the overexpression of the SREBP-1 gene of 1. goats, the SREBP-1 gene (1-1209 BP, n SREBP1) obtained from the goat was constructed into the P Adtrack-CMV shuttle vector. After the recombination of the skeleton carrier P Ad Easy-1, the HEK293 cells were transfected and the recombinant adenovirus of the high titer was amplified and amplified, and 48 of the primary mammary epithelial cells of the goat were infected. The gene m RNA level and protein level were significantly up-regulated, and the genes related to fatty acid synthesis were also caused by genes such as ACSL1, FABP3, ELOVL6, SCD1, ACSS2, ACLY, ACACA, FASN, IDH1, INSIG1, etc. The expression of SREBP-1 gene significantly up-regulated the triglyceride synthesis related genes LPIN1, DGAT1 expression and intracellular triglyceride content (P0.01), the content of C16:0 and C18:1 in cells increased significantly (P0.05), C16:1, C18:0 and C18:2 content decreased significantly (P0.05), but the content of long chain polyunsaturated fatty acids (such as C20:4 and impurities) in cells was not obvious. P0.05.2. goat SREBP-1 gene RNA interference design and synthesis of specific target to the SREBP-1 gene amino terminal active region of Si RNA, diluted after transfection of goat primary mammary epithelial cells, 48 h, SREBF1 and SREBP-1a gene expression significantly down 60% (P0.01), SREBP1 protein mature form expression significantly reduced the gene SCD1, ELOVL6, ACLY, ACSS2, IDH1, FABP3, ACSL1, SLC27A6, INSIG1, SCAP, PPARG and CPT1A. No significant change in expression (P0.05) the interference of.SREBP1 gene leads to a significant reduction in intracellular triglyceride content (P0.05). The content of intracellular cholesterol reduces the effect of.3.LXR alpha -SREBP1 pathway on fatty acid metabolism in goat mammary glands, using LXR alpha specific irritable agent T0901317 to treat 48 h of goat mammary epithelial cells, SREB The expression of P1 gene transcription level and protein level increased gradually, and the accumulation of SREBP-1 protein in the mature form of the nucleus increased. At the same time, the addition of T0901317 activates the activation of long chain fatty acids, translocation, desaturation, ab initio synthesis of fatty acids, and the synthesis of glycerol three ester, leading to SCL27A6, ACSL1, FABP3, SCD1, and so on. ELOVL6, ACSS2, ACLY, ACACA, FASN, IDH1, GPAM, LPIN1, DGAT1, DGAT2, PLIN2, PLIN3, etc. The content of intracellular C16:0 and C18:2 decreased, the content of C18:0 decreased significantly (P0.05), and the content of monounsaturated fatty acids (C16:1, C18:1, C20:1 and C22:1) increased significantly (P0.05), and the content of long chain polyunsaturated fatty acids decreased significantly (P0.01). The transcriptional regulation of.4. goat SREBP-1c genes was 2186. The 2012 bp. bioinformatics analysis of the upstream transcriptional initiation site found that the SREBP-1c promoter sequence contained two SREBP1 binding sites (SRE), two LXR binding sites (LXRE) and nuclear factor Y binding site (NF-Y) and Sp1 loci near two SRE sites. The activity of fluorescein enzyme activity analysis showed that LXR agonist T0901317 could be significantly higher The promoter activity of SREBP-1c was modulated (P0.05), while the Si RNA of the SREBP1 gene resulted in a significant downregulation of the SREBP-1c promoter activity (P0.01). The deletion mutation indicated that the core region of the SREBP-1c gene promoter was located at -395 bp~+1 BP. The mutant SRE and LXRE loci were lost to the original goat mammary epithelial cells. The results showed that the single or double mutation of the two SRE loci resulted in a significant downregulation of the basal activity of the SREBP-1c promoter (P0.01), while the mutation of the LXRE site had no significant downregulation to the promoter activity (P0.05). The single site LXRE was treated with T0901317. Or the mutation of the SRE site causes T0901317 to weaken the activation of SREBP-1c promoter, while the simultaneous mutation of the two LXRE loci causes the activation of T0901317 to SREBP-1c promoter to disappear. The results show that LXR A and N SREBP1 both play an important role in regulating the expression of SREBP-1c gene in goats, and the transcriptional regulation of.5. goat ACSS2 genes. The sequence of the 5'flanking sequence of the goat ACSS2 gene promoter was amplified by amplification, and the sequence analysis of the 1976 bp. promoter in the upstream of the transcriptional starting site showed that there was a transcription factor binding site of SREBP-1, LXR, NF-Y, Sp1 and STAT5 on the ACSS2 promoter. The promoter activity of the promoter (P0.05), and the LXR specific agonist T0901317 caused a significant increase in the activity of the ACSS2 promoter (P0.05). The deletion mutation found that the core region of the ACSS2 gene promoter is located in -515 bp~+1 BP and is in a conserved SRE site in the region. After a fixed point mutation of the SRE site, the activity of the ACSS2 promoter is shown to be active. Down regulation (P0.05). The results of chromatin immunoprecipitation experiment showed that SREBP1 protein was combined with the sequence of SRE components on ACSS2 promoter and directly regulated the effect of ACSS2 gene expression.6.ACSS2 and ACLY interference on the synthesis of fatty acids and triglycerides and synthesized Si RNA for goat ACSS2 and ACLY genes, which were transfected to the original goat. After 48 h of mammary epithelial cells, the expression of M RNA in ACSS2 and ACLY genes decreased by 70% (P0.05), while the gene expression of lipid metabolism related genes, ACACA, FASN, SCD1, ELOVL6, DGAT1, decreased, the accumulation of lipid droplets in cells decreased, and the content of triglycerides decreased. To sum up, SREBP1 gene is widely involved in regulating the expression of related genes in biological processes such as fatty acid biosynthesis, transport and triglyceride synthesis, increasing the content of monounsaturated fatty acids in cells and reducing the content of long chain polyunsaturated fatty acids.LXR a gene directly activates the expression of SREBP-1 gene and co regulates goat mammary fat. Fatty acid metabolism, and goat SREBP-1c gene is regulated by LXR alpha and its mature form of n SREBP1,.SREBP-1 gene can directly regulate the expression of ACSS2 gene at the transcriptional level, and affect the synthesis of fatty acids and triglycerides.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S827

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