本文選題：奶山羊 + SREBP1��； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：固醇調(diào)節(jié)元件結(jié)合蛋白-1(Sterol regulatory element binding protein-1,SREBP-1)是哺乳動物體內(nèi)重要的核轉(zhuǎn)錄因子之一,主要參與調(diào)控內(nèi)源的膽固醇、脂肪酸、磷脂及甘油三酯等的生物合成及代謝。在反芻動物乳腺組織中,SREBP-1通過激活乳脂合成及分泌過程中關(guān)鍵基因及酶的表達,促進脂肪酸的從頭合成過程,調(diào)控乳中有益脂肪酸的成分及含量,是乳脂合成的關(guān)鍵調(diào)控因子。羊奶含有豐富的短中鏈脂肪酸(C≤16),因此,通過奶山羊SREBP-1基因的功能研究解析乳腺上皮細胞脂肪酸代謝調(diào)控作用,對奶山羊育種及羊奶成分調(diào)控具有重要理論意義和實際應(yīng)用價值。本研究通過腺病毒介導(dǎo)的超表達技術(shù)及si RNA介導(dǎo)的干擾技術(shù)對SREBP-1基因的基本功能進行探討,并使用LXR-SREBP1通路的激動劑(T0901317)進行功能驗證;通過對山羊SREBP-1c及ACSS2基因啟動子的克隆及突變研究,探討山羊SREBP-1c基因及ACSS2基因的轉(zhuǎn)錄調(diào)控機制;通過對脂肪酸從頭合成過程中關(guān)鍵酶ACSS2及ACLY基因的干擾研究,驗證其對脂肪酸從頭合成及甘油三酯合成的影響。本研究的主要結(jié)果如下:1.山羊SREBP-1基因的過表達研究將克隆得到的山羊SREBP-1基因(1-1 209 bp,n SREBP1)構(gòu)建到p Adtrack-CMV穿梭載體中,與骨架載體p Ad Easy-1重組后,轉(zhuǎn)染HEK293細胞,包裝擴增出高滴度的重組腺病毒Ad-n SREBP1,感染山羊原代乳腺上皮細胞48 h后,SREBP-1基因m RNA水平及蛋白水平顯著上調(diào),同時導(dǎo)致脂肪酸合成代謝相關(guān)基因:ACSL1,FABP3,ELOVL6,SCD1,ACSS2,ACLY,ACACA,FASN,IDH1,INSIG1,NR1H3,PPARG等基因的表達量顯著上調(diào)(P0.05),而SREBP-1蛋白成熟裂解過程的護送蛋白SCAP的表達量則顯著下調(diào)(P0.05)。同時,過表達SREBP-1基因顯著上調(diào)甘油三酯合成相關(guān)基因LPIN1、DGAT1的表達量及細胞內(nèi)甘油三酯的含量(P0.01),細胞內(nèi)C16:0及C18:1的含量顯著增加(P0.05),C16:1,C18:0及C18:2的含量顯著減少(P0.05),而細胞內(nèi)長鏈多不飽和脂肪酸(如C20:4及C22:6)的含量無明顯變化(P0.05)。2.山羊SREBP-1基因的RNA干擾設(shè)計并合成特異靶向SREBP-1基因氨基末端活性區(qū)域的si RNA,稀釋后轉(zhuǎn)染山羊原代乳腺上皮細胞,48 h后,SREBF1及SREBP-1a基因表達量顯著下調(diào)60%以上(P0.01),SREBP1蛋白成熟形式表達量顯著減少。SREBP1基因的干擾導(dǎo)致細胞內(nèi)脂肪酸合成、轉(zhuǎn)運相關(guān)基因及脂代謝調(diào)控因子(SCD1,ELOVL6,ACLY,ACSS2,IDH1,FABP3,ACSL1,SLC27A6,INSIG1,SCAP,PPARG及CPT1A)的表達量顯著下調(diào)(P0.05),PLIN3,XDH,PPARA及ACOX的表達量顯著上調(diào)(P0.05),ACACA,FASN,CD36,GPAM,DGAT1,PLIN2,NR1H3,ATGL,HSL等基因的表達量無顯著變化(P0.05)。SREBP1基因的干擾導(dǎo)致細胞內(nèi)甘油三酯的含量顯著下調(diào)(P0.05),細胞內(nèi)膽固醇的含量下調(diào)。3.LXRα-SREBP1通路激活對山羊乳腺脂肪酸代謝的影響使用不同濃度LXRα特異性激動劑T0901317處理山羊乳腺上皮細胞48 h后,SREBP1基因轉(zhuǎn)錄水平及蛋白水平的表達量均呈梯度上升趨勢,并且細胞核內(nèi)成熟形式的SREBP-1蛋白積累增加。同時,T0901317的添加激活細胞內(nèi)長鏈脂肪酸激活、轉(zhuǎn)運、去飽和,脂肪酸從頭合成,及甘油三酯合成等生理過程,導(dǎo)致SCL27A6,ACSL1,FABP3,SCD1,ELOVL6,ACSS2,ACLY,ACACA,FASN,IDH1,GPAM,LPIN1,DGAT1,DGAT2,PLIN2,PLIN3,ATGL,XDH等基因的表達量顯著上調(diào)(P0.05),同時脂代謝調(diào)控因子INSIG1,SCAP,PPARA及PPARG的表達量也顯著上調(diào)(P0.05)。LXRα-SREBP1通路的激活導(dǎo)致細胞內(nèi)脂滴數(shù)量及甘油三酯含量均顯著增加,細胞內(nèi)C16:0及C18:2含量降低,C18:0的含量顯著減少(P0.05),單不飽和脂肪酸(C16:1,C18:1,C20:1及C22:1)的含量顯著增加(P0.05),長鏈多不飽和脂肪酸的含量顯著降低(P0.01)。4.山羊SREBP-1c基因的轉(zhuǎn)錄調(diào)控作用克隆得到山羊SREBP-1c基因5'側(cè)翼序列2 186 bp,含轉(zhuǎn)錄起始位點上游2 012 bp。生物信息學(xué)分析發(fā)現(xiàn),SREBP-1c啟動子序列含有兩個SREBP1結(jié)合位點(SRE),兩個LXR結(jié)合位點(LXRE),并且在兩個SRE位點附近含有核因子Y結(jié)合位點(NF-Y)及Sp1位點。熒光素酶活性分析表明,LXR的激動劑T0901317可以顯著上調(diào)SREBP-1c的啟動子活性(P0.05),而SREBP1基因的si RNA導(dǎo)致SREBP-1c啟動子活性顯著下調(diào)(P0.01)。缺失突變結(jié)果表明,SREBP-1c基因啟動子核心區(qū)域位于-395 bp~+1 bp;并且,當(dāng)SREBP-1c啟動子由-395 bp缺失到-86 bp時,T0901317對SREBP-1c基因啟動子的激活作用消失。定點突變SRE及LXRE位點后轉(zhuǎn)染山羊原代乳腺上皮細胞,結(jié)果表明:兩個SRE位點的單突變或者雙突變均導(dǎo)致SREBP-1c啟動子基礎(chǔ)活性顯著下調(diào)(P0.01),而LXRE位點的突變對啟動子活性無明顯下調(diào)作用(P0.05)。使用T0901317處理后,單一位點的LXRE或者SRE位點的突變均導(dǎo)致T0901317對SREBP-1c啟動子的激活作用減弱,而兩個LXRE位點的同時突變則導(dǎo)致T0901317對SREBP-1c啟動子的激活作用消失。結(jié)果表明,LXRα及n SREBP1均對山羊SREBP-1c基因的表達有重要的調(diào)控作用。5.山羊ACSS2基因的轉(zhuǎn)錄調(diào)控作用擴增得到山羊ACSS2基因啟動子5'側(cè)翼序列2 359 bp,含轉(zhuǎn)錄起始位點上游1 976 bp。啟動子序列分析結(jié)果表明,ACSS2啟動子上存在SREBP-1,LXR,NF-Y,Sp1和STAT5等轉(zhuǎn)錄因子結(jié)合位點。啟動子熒光素酶活性分析發(fā)現(xiàn),SREBP1基因的si RNA顯著下調(diào)ACSS2基因啟動子活性(P0.05),而LXR的特異性激動劑T0901317導(dǎo)致ACSS2基因啟動子活性顯著升高(P0.05)。缺失突變發(fā)現(xiàn),ACSS2基因啟動子核心區(qū)域位于-515 bp~+1 bp,并且在該區(qū)域內(nèi)存在一個保守的SRE位點。對SRE位點進行定點突變后,導(dǎo)致ACSS2啟動子活性顯著下調(diào)(P0.05)。染色質(zhì)免疫共沉淀實驗結(jié)果表明,SREBP1蛋白與ACSS2啟動子上的SRE元件序列結(jié)合,直接調(diào)控ACSS2基因的表達。6.ACSS2及ACLY共同干擾對脂肪酸及甘油三酯合成的影響設(shè)計并合成特異靶向山羊ACSS2及ACLY基因的si RNA,轉(zhuǎn)染山羊原代乳腺上皮細胞48 h后,ACSS2及ACLY基因m RNA表達量顯著下調(diào)70%左右(P0.05),同時,細胞內(nèi)脂代謝相關(guān)基因ACACA,FASN,SCD1,ELOVL6,DGAT1,DGAT2,AGPAT6,FABP3,CD36,PPARA,ACOX,CPT1A,PLIN2,PLIN3等基因的表達量顯著下調(diào)(P0.05),細胞內(nèi)脂滴積累減少,甘油三酯含量降低。綜上所述,SREBP1基因廣泛參與調(diào)控脂肪酸生物合成、轉(zhuǎn)運及甘油三酯合成等生物過程中相關(guān)基因的表達,增加細胞內(nèi)單不飽和脂肪酸的含量,降低長鏈多不飽和脂肪酸的含量。LXRα基因可直接激活SREBP-1基因的表達,共同調(diào)控山羊乳腺脂肪酸代謝,并且山羊SREBP-1c基因同時受到LXRα及其成熟形式n SREBP1的調(diào)控。SREBP-1基因可在轉(zhuǎn)錄水平直接調(diào)控ACSS2基因的表達,影響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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S827

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