UCP3基因在巴马猪和藏猪脂肪组织中的表达和甲基化分析

doi:10.16431/j.cnki.1671-7236.2019.12.020

摘要/Abstract

摘要： 为探究解偶联蛋白3（uncoupling protein 3，UCP3）基因在巴马猪和藏猪皮下脂肪组织中的表达和甲基化水平，试验采用实时荧光定量PCR技术检测UCP3基因在巴马猪和藏猪皮下脂肪组织中的mRNA表达水平；针对猪UCP3基因启动子区域（-3 580~+920 bp），利用在线软件MethPrimer对该区域进行CpG岛预测，并采用亚硫酸氢盐测序法（bisulfite sequencing PCR，BSP）检测其甲基化水平，探究UCP3基因甲基化水平在巴马猪和藏猪中的差异。结果显示，巴马猪皮下脂肪组织UCP3基因表达量显著高于藏猪（P<0.05）；在UCP3基因启动子区预测到3个CpG甲基化岛，分别是CpG island1（-3 171~-2 928 bp）、CpG island2（-154~-2 bp）和CpG island3（+648~+806 bp），其中CpG island1和CpG island3的甲基化水平在巴马猪和藏猪中差异较小，而藏猪CpG island2的甲基化水平（42.61%）高于巴马猪（24.49%）。本研究绘制了2个猪种CpG island2甲基化水平的黑白点图，其中CpG位点为4、8、9、10、11、12、15，藏猪甲基化频率分别比巴马猪高28.26%、17.39%、26.09%、26.09%、26.09%、23.91%和34.78%。在CpG island2处预测到3个转录因子结合位点（SP2、PPARγ和EGR1）。结果表明，巴马猪和藏猪皮下脂肪组织中UCP3基因mRNA水平的表达差异可能是由于CpG island2的甲基化水平不同所导致，藏猪DNA甲基化水平在一定程度上阻碍了转录因子与启动子调控区域的结合，从而抑制了UCP3基因的表达。

关键词: 巴马猪; 藏猪; UCP3基因; 脂肪组织; 甲基化

Abstract: The aim of this study was to investigate the expression and methylation levels of the uncoupling protein 3 (UCP3) in subcutaneous adipose tissue of Bama and Tibetan pigs.The expression of UCP3 gene at mRNA level was detected by Real-time quantitative PCR method.CpG islands were predicted for the sequence in pig UCP3 gene promoter region (-3 580 to +920 bp) using online software MethPrimer.The difference methylation level of UCP3 gene in Bama and Tibetan pigs was detected by bisulfite sequencing PCR (BSP).The results showed that the expression of UCP3 gene in subcutaneous adipose tissue of Bama pigs was significantly higher than that of Tibetan pigs (P<0.05).Three CpG methylation islands were predicted in the promoter region of UCP3 gene,which were recorded as CpG island1 (-3 171 to -2 928 bp),CpG island2 (-154 to -2 bp) and CpG island3 (+648 to +806 bp).The methylation levels of CpG island1 and CpG island3 were less different in Bama and Tibetan pigs,while the methylation level of CpG island2 (42.61%) was significantly higher in Tibetan pigs than that in Bama pigs (24.49%).A black and white dot maps were built based on the CpG island2 methylation of Bama and Tibetan pigs,the methylation frequency of CpG sites 4,8,9,10,11,12 and 15 in Tibetan pigs were 28.26%,17.39%,26.09%,26.09%,26.09%,23.91% and 34.78% higher than that in Bama pigs,respectively.In addition,three transcription factor binding sites (SP2,PPARγ and EGR1) were predicted at CpG island2.The results indicated that the differential expression of UCP3 gene in subcutaneous adipose tissue of Bama and Tibetan pigs might be caused by the different methylation levels of CpG island2.The higher DNA methylation of adipose tissue in Tibetan pigs might inhibit the expression of UCP3 gene at mRNA level by inhibiting the binding of transcription factors to promoter regulatory regions.

Key words: Bama pigs; Tibetan pigs; UCP3 gene; adipose tissue; methylation

中图分类号:

范一萍, 王彦芳, 陶聪. UCP3基因在巴马猪和藏猪脂肪组织中的表达和甲基化分析[J]. 中国畜牧兽医, 2019, 46(12): 3627-3634.

FAN Yiping, WANG Yanfang, TAO Cong. Expression and Methylation Analysis of UCP3 Gene in Adipose Tissue of Bama and Tibetan Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(12): 3627-3634.

参考文献

[1] 温佳,王一民,葛静,等.动物白色脂肪组织棕色化的调控机制[J].家畜生态学报,2018,39(8):8-12. WEN J,WANG Y M,GE J,et al.Regulatory mechanism of white adipose tissue browning in animal[J].Journal of Domestic Animal Ecology,2018,39(8):8-12.(in Chinese)
[2] 姚旋,张颖,单仕芳,等.褐色脂肪组织研究的最新进展和科学意义[J].中国细胞生物学学报,2011,33(3):227-236. YAO X,ZHANG Y,SHAN S F,et al.Recent progress in the study of brown adipose tissue and its scientific significance[J].Chinese Jouranl of Cell Biology,2011,33(3):227-236.(in Chinese)
[3] 王亚君.脂肪特异性ATP7A敲除小鼠抵抗饮食诱导的肥胖[D].北京:中国农业科学院,2017. WANG Y J.Adipocyte-specific ATP7A knockout mice are resistant to diet-induced obesity[D].Beijing:Chinese Academy of Agricultural Sciences,2017.(in Chinese)
[4] FARMER S R.Molecular determinants of brown adipocyte formation and function[J].Genes & Development,2008,22(10):1269-1275.
[5] KLINGENBERG M,HUANG S G.Structure and function of the uncoupling protein from brown adipose tissue[J].Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids,1999,1415(2):271-296.
[6] FENZL A,KIEFER F W.Brown adipose tissue and thermogenesis[J].Hormone Molecular Biology and Clinical Investigation,2014,19(1):25-37.
[7] ANDRIY F,LISHKO P V,YURIY K.Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria[J].Cell,2012,151(2):400-413.
[8] HARMS M,SEALE P.Brown and beige fat:Development,function and therapeutic potential[J].Nature Medicine,2013,19(10):1252-1263.
[9] WANG W,SEALE P.Control of brown and beige fat development[J].Nature Reviews Molecular Cell Biology,2016,17(11):691-702.
[10] OELKRUG R,POLYMEROPOULOS E T,JASTROCH M.Brown adipose tissue:Physiological function and evolutionary significance[J].Journal of Comparative Physiology B,2015,185(6):587-606.
[11] BERG F,GUSTAFSON U,ANDERSSON L.The uncoupling protein 1 gene (UCP1) is disrupted in the pig lineage:A genetic explanation for poor thermoregulation in piglets[J].PLoS Genetics,2006,2(8):e129.
[12] LIN R,LIN W,CHEN Q,et al.Gene expression and promoter methylation of porcine uncoupling protein 3 gene[J].Asian-Australasian Journal of Animal Sciences,2018,32(2):170-175.
[13] 涂荣剑.猪解耦联蛋白基因3(UCP3)的序列及多态性研究[D].武汉:华中农业大学,2003. TU R J.Reserch on sequence and polymorphism of porcine uncoupling protein gene 3[D].Wuhan:Huazhong Agricultural University,2003.(in Chinese)
[14] MOZO J,FERRY G,STUDENY A,et al.Expression of UCP3 in CHO cells does not cause uncoupling,but controls mitochondrial activity in the presence of glucose[J].Biochemical Journal,2006,393(1):431-439.
[15] PATRICK S.The role of uncoupling protein 3 in fatty acid metabolism:Protection against lipotoxicity[J].Proceedings of the Nutrition Society,2004,63(2):287-292.
[16] ALISON M,BOS P M,LITTEN J C,et al.Differential effects of thyroid hormone manipulation and beta adrenoceptor agonist administration on uncoupling protein mRNA abundance in adipose tissue and thermoregulation in neonatal pigs[J].Organogenesis,2008,4(3):182-187.
[17] CLAPHAM J C,ARCH J R,CHAPMAN H,et al.Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean[J].Nature,2000,406(6794):415-418.
[18] BARTZ M,NOWACKA-WOSZUK J,BARTZ M,et al.Association studies on the porcine RETN,UCP1,UCP3 and ADRB3 genes polymorphism with fatness traits[J].Meat Science,2009,83(3):551-554.
[19] BRAND M D,ESTEVES T C.Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3[J].Cell Metabolism,2005,2(2):85-93.
[20] LIN J,CAO C,TAO C,et al.Cold adaptation in pigs depends on UCP3 in beige adipocytes[J].Journal of Molecular Cell Biology,2017,9(5):364-375.
[21] 张永红.猪UCP3基因多态性与生长性状相关性的研究[D].长沙:湖南农业大学,2005. ZHANG Y H.Study on effect of UCP3 gene polymorphism on growth traits in pigs[D].Changsha:Hunan Agricultural University,2005.(in Chinese)
[22] SCHRAUWEN P,HOEKS J,HESSELINK M K.Putative function and physiological relevance of the mitochondrial uncoupling protein-3:Involvement in fatty acid metabolism[J].Progress in Lipid Research,2006,45(1):17-41.
[23] 张凯,顾以韧,刘艾晶,等.长白猪和梅山猪脂肪中脂肪沉积相关基因的表达差异[J].畜牧兽医学报,2010,41(2):129-134. ZHANG K,GU Y R,LIU A J,et al.Expression changes of genes eelated to adipose deposition during postnatal development of backfat in Landrace and Meishan pigs[J].Acta Veterinaria et Zootechnica Sinica,2010,41(2):129-134.(in Chinese)
[24] 杨芳,龙开旭,方程,等.陆川猪与杜洛克猪正反交F1代UCP3基因表达差异研究[J].中国畜牧兽医,2016,43(3):798-804. YANG F,LONG K X,FANG C,et al.Study on differences of UCP3 gene expression of reciprocal crosses F1 with Luchuan pig and Duroc pig[J].China Animal Husbandry & Veterinatry Medicine,2016,43(3):798-804.(in Chinese)
[25] CASTELLO D C,INDIAS I M,ALCOHOLADO L S,et al.Altered adipose tissue DNA methylation status in metabolic syndrome:Relationships between global DNA methylation and specific methylation at adipogenic,lipid metabolism and inflammatory candidate genes and metabolic variables[J].Journal of Clinical Medicine,2019,8(1):E87.
[26] LAIRD P W.Principles and challenges of genome-wide DNA methylation analysis[J].Nature Reviews Genetics,2010,11(3):191-203.
[27] HOVESTADT V,JONES D T,PICELLI S,et al.Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing[J].Nature,2014,510(7506):537-541.