山羊DGAT1基因的克隆及对前体脂肪细胞脂质沉积的调控作用研究

doi:10.11843/j.issn.0366-6964.2022.01.008

Abstract

Abstract: The aim of the study was to clone the sequence of DGAT1 gene, clarify its expression pattern in different tissues of goat, and further reveal the role of DGAT1 gene in regulating lipid metabolism in goat intramuscular preadipocytes. In this study, seven healthy 10-month-old Jianzhou male goats were used as experimental animals. The sequence of DGAT1 gene was cloned by RT-PCR and analyzed using bioinformatics. The relative expression level of DGAT1 in different tissues of goat were determined by real-time quantitative PCR (RT-qPCR). The eukaryotic expression vector pcDNA3.1-DGAT1 was constructed by double enzyme digestion and transfected into goat intramuscular preadipocytes. RT-qPCR was used for detecting the expression of DGAT1 and lipid metabolism-related genes. The effect of overexpression of DGAT1 on lipid droplet formation was observed by Oil red O staining method, and the GPO-Trinder enzyme reaction was used to measure triglyceride content. A length of 1 651 bp of DGAT1 gene was cloned (GenBank accession number： MT221183), including 125 bp 5'UTR, 1 470 bp CDS, and 56 bp 3'UTR, encoding 489 amino acid residues. The goat DGAT1 gene had the highest expression in small intestine and the lowest in spleen. RT-qPCR results showed that DGAT1 was extremely significantly overexpressed in cells (P<0.01) by pcDNA3.1-DGAT1 treatment, as a results, the expression of GPAM (P<0.05), ADRP (P<0.01) and ACOX1 (P<0.01) were significantly up-regulated, while the expression of AGPAT6 (P<0.05), MLYCD (P<0.01) and HSL (P<0.01) were significantly down-regulated. Oil red O staining results showed that the lipid droplet accumulation after overexpression of DGAT1 was significantly (P<0.01) increased compared with that in the control group, as well as the results of triglyceride determination (P<0.01) in intramuscular adipocytes in goats. This study successfully obtained the goat DGAT1 gene CDS region sequence and constructed the pcDNA3.1-DGAT1 eukaryotic expression vector, overexpression of DGAT1 could significantly promote lipid deposition of intramuscular preadipocytes in goats and significantly affect the expression of lipid metabolism-related genes. These results provide important data for further clarifying the mechanism of DGAT1 in regulating intramuscular lipid metabolism in goats.

Key words: goat, DGAT1, overexpression, intramuscular preadipocytes, triglycerides

CLC Number:

S827.2

YANG Changheng, LI Qi, HUANG Wei, LIN Yaqiu, WANG Yong, XIANG Hua, ZHU Jiangjiang. Cloning of Goat DGAT1 Gene and Its Regulation on Lipid Deposition in Preadipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 76-87.

References 39

[1]	WOOD J D,ENSER M,FISHER A V,et al.Fat deposition,fatty acid composition and meat quality:A review[J].Meat Sci,2008,78(4): 343-358.
[2]	TALPUR F N,BHANGER M I,SHERAZI S T H.Intramuscular fatty acid profile of longissimus dorsi and semitendinosus muscle from Pateri goats fed under traditional feeding system of Sindh,Pakistan[J].Meat Sci,2008,80(3):819-822.
[3]	HWANG Y H,LEE S J,LEE E Y,et al.Effects of carcass weight increase on meat quality and sensory properties of pork loin[J].J Anim Sci Technol,2020,62(5):753-760.
[4]	YU H B,ZHAO Y L,IQBAL A,et al.Effects of polymorphism of the GPAM gene on milk quality traits and its relation to triglyceride metabolism in bovine mammary epithelial cells of dairy cattle[J].Arch Anim Breed,2021,64(1):35-44.
[5]	龙小娟.牛AGPAT6基因遗传多态性研究及与脂代谢相关功能的验证[D].长春:吉林大学,2016.LONG X J.Polymorphism detection and function verifiction with lipid metabolism of AGPAT6 gene in cattle[D].Changchun: Jilin University,2016.(in Chinese)
[6]	余康.奶山羊ADRP基因对GMEC脂滴的影响及其分子机制的初步研究[D].杨凌:西北农林科技大学,2014.YU K.Effects of ADRP gene of dairy goat to lipid droplets in GMEC (Goat Mammary Gland Epithelial Cells) and its preliminary molecular mechanism exploration[D].Yangling:Northwest A&F University,2014.(in Chinese)
[7]	SCHREIBER R,XIE H,SCHWEIGER M.Of mice and men:The physiological role of adipose triglyceride lipase (ATGL)[J].Biochim Biophys Acta Mol Cell Biol Lipids,2019,1864(6):880-899.
[8]	KRAEMER F B,SHEN W J.Hormone-sensitive lipase knockouts[J].Nutr Metab (Lond),2006,3(1):12.
[9]	BREJCHOVA K,RADNER F P W,BALAS L,et al.Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides[J].Proc Natl Acad Sci U S A,2021,118(2):e2020999118.
[10]	WEISS S B,KENNEDY E P,KIYASU J Y.The enzymatic synthesis of triglycerides[J].J Biol Chem,1960,235(1):40-44.
[11]	CASES S,SMITH S J,ZHENG Y W,et al.Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase,a key enzyme in triacylglycerol synthesis[J].Proc Natl Acad Sci U S A,1998,95(22):13018-13023.
[12]	OELKERS P,BEHARI A,CROMLEY D,et al.Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase-related enzymes[J].J Biol Chem,1998,273(41):26765-26771.
[13]	GRISART B,COPPIETERS W,FARNIR F,et al.Positional candidate cloning of a QTL in dairy cattle:identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition[J].Genome Res,2002,12(2):222-231.
[14]	NONNEMAN D,ROHRER G A.Linkage mapping of porcine DGAT1 to a region of chromosome 4 that contains QTL for growth and fatness[J].Anim Genet,2002,33(6):472-473.
[15]	CHEN H C,FARESE JR R V.Inhibition of triglyceride synthesis as a treatment strategy for obesity:lessons from DGAT1-deficient mice[J].Arterioscler Thromb Vasc Biol,2005,25(3):482-486.
[16]	CHEN H C,STONE S J,ZHOU P,et al.Dissociation of obesity and impaired glucose disposal in mice overexpressing acyl coenzyme a:diacylglycerol acyltransferase 1 in white adipose tissue[J].Diabetes,2002,51(11):3189-3195.
[17]	YU T,WU C D,SHIH N Y,et al.Discovery of dimethyl pent-4-ynoic acid derivatives,as potent and orally bioavailable DGAT1 inhibitors that suppress body weight in diet-induced mouse obesity model[J].Bioorg Med Chem Lett,2018,28(10):1686-1692.
[18]	HUNG Y H,BUHMAN K K.DGAT1 deficiency disrupts lysosome function in enterocytes during dietary fat absorption[J].Biochim Biophys Acta Mol Cell Biol Lipids,2019,1864(4):587-595.
[19]	YAMAZAKI T,SASAKI E,KAKINUMA C,et al.Increased very low density lipoprotein secretion and gonadal fat mass in mice overexpressing liver DGAT1[J].J Biol Chem,2005,280(22):21506-21514.
[20]	MILLAR J S,STONE S J,TIETGE U J F,et al.Short-term overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not VLDL triglyceride or apoB production[J].J Lipid Res,2006,47(10):2297-2305.
[21]	ROORDA B D,HESSELINK M K C,SCHAART G,et al.DGAT1 overexpression in muscle by in vivo DNA electroporation increases intramyocellular lipid content[J].J Lipid Res,2005,46(2):230-236.
[22]	LIU L,SHI X J,CHOI C S,et al.Paradoxical coupling of triglyceride synthesis and fatty acid oxidation in skeletal muscle overexpressing DGAT1[J].Diabetes,2009,58(11):2516-2524.
[23]	ROE N D,HANDZLIK M K,LI T,et al.The role of diacylglycerol acyltransferase (DGAT) 1 and 2 in cardiac metabolism and function[J].Sci Rep,2018,8(1):4983.
[24]	芦春艳.奶牛乳腺上皮细胞系的建立及DGAT1基因功能的验证[D].长春:吉林大学,2012.LU C Y.Establishment of mammary epithelial cell lines and RNA interference-mediated knockdown of DGAT1 decreases TG content of cow[D].Changchun:Jilin University,2012.(in Chinese)
[25]	毛永江,陈仁金,陈莹,等.南方地区中国荷斯坦牛DGAT1基因多态性与泌乳性状的关联分析[J].中国农业科学,2010,43(14):2990-2995.MAO Y J,CHEN R J,CHEN Y,et al.Analysis of association between DGAT1 gene and milking traits in Chinese Holstein in the south of China[J].Scientia Agricultura Sinica,2010,43(14):2990-2995.(in Chinese)
[26]	LI F Y,CAI C C,QU K X,et al.DGAT1 K232A polymorphism is associated with milk production traits in Chinese cattle[J].Anim Biotechnol,2021,32(4):427-431.
[27]	SUN Y T,LUO J,ZHU J J,et al.Effect of short-chain fatty acids on triacylglycerol accumulation,lipid droplet formation and lipogenic gene expression in goat mammary epithelial cells[J].Anim Sci J,2016,87(2):242-249.
[28]	林森,林亚秋,朱江江,等.Wnt10b对山羊前体脂肪细胞分化相关基因表达的影响[J].畜牧兽医学报,2018,49(4):685-692.LIN S,LIN Y Q,ZHU J J,et al.Effect of Wnt10b on the expression of precursor adipocytes differentiation related genes in goat[J].Acta Veterinaria et Zootechnica Sinica,2018,49(4):685-692.(in Chinese)
[29]	许晴,李倩,王永,等.过表达FGF10促进山羊皮下前体脂肪细胞分化[J].畜牧兽医学报,2019,50(10):1972-1984.XU Q,LI Q,WANG Y,et al.Overexpression of FGF10 promotes the differentiation of subcutaneous preadipocyte in goat[J].Acta Veterinaria et Zootechnica Sinica,2019,50(10):1972-1984.(in Chinese)
[30]	朱江江.FASN基因对奶山羊乳腺脂肪酸代谢的调控作用研究[D].杨凌:西北农林科技大学,2015.ZHU J J.Regulation of FASN gene in fatty acid metabolism of goat mammary gland[D].Yangling:Northwest A&F University, 2015.(in Chinese)
[31]	许晴,林森,朱江江,等.山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析[J].畜牧兽医学报,2018, 49(5): 907-918.XU Q,LIN S,ZHU J J,et al.The expression stability analysis of reference genes in the process of goat intramuscular preadipocytes differentiation in goat[J].Acta Veterinaria et Zootechnica Sinica,2018,49(5):907-918.(in Chinese)
[32]	ANGIOLILLO A,AMILLS M,URRUTIA B,et al.Identification of a single nucleotide polymorphism at intron 16 of the caprine Acyl-Coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) gene[J].J Dairy Res,2007,74(1):47-51.
[33]	CAO H P.Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms[J].BMC Res Notes,2011, 4(1):249.
[34]	LIU Q,SILOTO R M P,LEHNER R,et al.Acyl-CoA:diacylglycerol acyltransferase:molecular biology,biochemistry and biotechnology[J]. Prog Lipid Res,2012,51(4):350-377.
[35]	CHITRAJU C,MEJHERT N,HAAS J T,et al.Triglyceride synthesis by DGAT1 protects adipocytes from lipid-induced ER stress during lipolysis[J].Cell Metab,2017,26(2):407-418.
[36]	KUERSCHNER L,MOESSINGER C,THIELE C.Imaging of lipid biosynthesis:how a neutral lipid enters lipid droplets[J].Traffic, 2008,9(3):338-352.
[37]	YEN C L E,STONE S J,KOLIWA S,et al.Thematic review series:glycerolipids.DGAT enzymes and triacylglycerol biosynthesis[J].J Lipid Res,2008,49(11):2283-2301.
[38]	WANG L,QIAN H W,NIAN Y,et al.Structure and mechanism of human diacylglycerol O-acyltransferase 1[J].Nature,2020, 581(7808):329-332.
[39]	KASAPKARA C S,CIVELEKÜREY B,CEYLAN A C,et al.Malonyl coenzyme A decarboxylase deficiency with a novel mutation[J].Cardiol Young,2021,31(9):1535-1537.

Cloning of Goat DGAT1 Gene and Its Regulation on Lipid Deposition in Preadipocytes

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