绵羊UCP4基因编码序列的克隆和mRNA表达研究

doi:10.3864/j.issn.0578-1752.2013.10.017

摘要/Abstract

摘要： 【目的】克隆绵羊UCP4基因的编码序列（CDS），分析CDS及其编码蛋白结构特点，并探讨其mRNA的发育性表达规律，以期为该基因的结构和功能研究奠定理论基础。【方法】利用RT-PCR技术从绵羊大脑组织中扩增出该基因的编码区序列，运用生物信息学方法分析UCP4蛋白的理化性质和结构特点。利用实时荧光定量PCR技术，对两个具有显著尾型差异的绵羊品种（广灵大尾羊和小尾寒羊）2、4、6、8、10和12月龄共计96个个体的8种组织（大脑、小脑、下丘脑、垂体、皮下脂肪、肾周脂肪、肠系膜脂肪和尾部脂肪）进行mRNA表达研究。【结果】绵羊UCP4基因CDS区长972 bp，编码323个氨基酸，分子量为35.73 kDa，等电点为9.43。二级结构中α螺旋、β折叠股和环分别占56.04%、7.12%和36.84%。该蛋白为跨膜蛋白，无信号肽，但有2个糖基化位点和15个潜在的磷酸化位点。UCP4 mRNA在脑组织和脂肪组织中均有表达，但高表达于脑组织中。品种、月龄和组织对UCP4 mRNA表达均有显著影响。【结论】获得了绵羊UCP4 基因的CDS全序列，揭示了其mRNA的表达特征及其影响因素，对进一步研究该基因的结构及其与能量代谢的关系具有重要科学意义。

关键词: 绵羊 , UCP4基因 , 编码序列 , mRNA表达

Abstract: 【Objective】This study aims to clone the coding sequence (CDS) of ovine UCP4 gene, to analyze the CDS and its coding protein structures, and to profile the ontogenetic mRNA expression so that to lay a theoretical foundation for future research on the structure and function of this gene. 【Method】The CDS was amplified from ovine cerebrum tissue by reverse transcription- polymerase chain reaction (RT-PCR). Physicochemical features and structural characteristics of ovine UCP4 were analyzed by bioinformatics methods. Ninety-six animals from two sheep breeds with significant differences in tail types (Guangling Large Tailed- and Small Tailed-Han Sheep) were used to study the ontogenetic mRNA expression by real-time quantitative PCR in eight tissues (cerebrum, cerebellum, hypothalamus, pituitary, and subcutaneous fat, perirenal fat, mesenteric fat and tail fat) from 2-to 12-month old age at two-month interval. 【Result】The sequencing data showed that the length of complete CDS was 972 bp encoding a protein of 323 amino acids. The isoelectric point (pI) of the protein was 9.43, and the molecular weight was 35.73 kDa. The percentages of helix, strand and loop were 56.04%, 7.12% and 36.84%, respectively, in the secondary structure. This transmembrane protein had no signal peptide but with two predicted glycosylation sites and 15 potential phosphorylation sites. The UCP4 mRNA expressed in all these tissues, but overexpressed in brain tissue other than in adipose tissue. Generally, breed, tissue and the months of age had significant influences on the UCP4 mRNA expression. 【Conclusion】 The obtained whole CDS of ovine UCP4 gene, expression profile and its limiting factors are of scientific significance to further studies on the relationship between the gene structure and energy metabolism.

Key words: sheep , UCP4 gene , coding sequence , mRNA expression

周沙沙, 刘宝凤, 魏琳琳, 王景霖, 刘建华, 梁琛, 乔利英, 刘文忠. 绵羊UCP4基因编码序列的克隆和mRNA表达研究[J]. 中国农业科学, 2013, 46(10): 2110-2118.

ZHOU Sha-Sha, LIU Bao-Feng, WEI Lin-Lin, WANG Jing-Lin, LIU Jian-Hua, LIANG Chen, QIAO Li-Ying, LIU Wen-Zhong. Study on the Cloning of Coding Sequence and Ontogenetic mRNA Expression of Ovine UCP4 Gene[J]. Scientia Agricultura Sinica, 2013, 46(10): 2110-2118.

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参考文献

[1]Brand M D, Chien L F, Ainscow E K, Rolfe D F, Porter R K. The cause and functions of mitochondrial proton leak. Biochemica et Biophysica Acta, 1994, 1187: 132-139.

[2]Ricquier D, Bouillaud F. Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance. Journal of Physiology, 2000, 529: 3-10.

[3]Yuan Y N, Liu W Z, Liu J H, Qiao L Y, Wu J L. Cloning and ontogenetic expression of the uncoupling protein 1 gene (UCP1) in sheep. Journal of Applied Genetics, 2012, 53: 203-212.

[4]Tian X Y, Wong W T, Xu A, Lu Y, Zhang Y, Wang L, Cheang W S, Wang Y, Yao X, Huang Y. Uncoupling protein-2 protects endothelial function in diet-induced obese mice. Circulation Research, 2012, 110: 1211-1216.

[5]Millet L, Vidal H, Larrouy D, Andreelli F, Laville M, Langin D. mRNA expression of the long and short forms of uncoupling Protein-3 in obese and lean humans. Diabetologia, 1998, 41: 829-832.

[6]Mao W, Yu X X, Zhong A, Li W, Brush J, Sherwood S W, Adams S H, Pan G. UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells. FEBS Letters, 1999, 443: 326-330.

[7]Ramsden D B, Ho P W, Ho J W, Liu H F, So D H, Tse H M, Chan K H, Ho S L. Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction. Brain and Behavior, 2012, 2: 468-478.

[8]Ho P W, Ho J W, Tse H M, So D H, Yiu D C, Liu H F, Chan K H, Kung M H, Ramsden D B, Ho S L. Uncoupling protein-4 (UCP4) increases ATP supply by interacting with mitochondrial Complex II in neuroblastoma cells. PLoS One, 2012, 7(2): e32810.

[9]Chu A C, Ho P W, Kwok K H, Ho J W, Chan K H, Liu H F, Kung M H, Ramsden D B, Ho S L. Mitochondrial UCP4 attenuates MPP+- and dopamine-induced oxidative stress, mitochondrial depolarization, and ATP deficiency in neurons and is interlinked with UCP2 expression. Free Radical Biology and Medicine, 2009, 46: 810-820.

[10]Yu X X, Mao W, Zhong A, Schow P, Brush J, Sherwood S W, Adams S H, Pan G. Characterization of novel UCP5/BMCP1 isoforms and differential regulation of UCP4 and UCP5 expression through dietary or temperature manipulation. Journal of the Federation of American Societies for Experimental Biology, 2000, 149: 1611-1618.

[11]Lengacher S, Magistretti P J, Pellerin L. Quantitative RT-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle. Journal of Cerebral Blood Flow & Metabolism, 2004, 24: 780-788.

[12]Gao C L, Ni Y H, Liu G, Chen X H, Ji C B, Qin D N, Kou C Z, Zhu C, Zhang C M, Xia Z K, Guo X R. UCP4 overexpression improves fatty acid oxidation and insulin sensitivity in L6 myocytes. Journal of Bioenergetics and Biomembranes, 2011, 43: 109-118.

[13]Zhang M, Wang B, Ni Y H, Liu F, Fei L, Pan X Q, Guo M, Chen R H, Guo X R. Overexpression of uncoupling protein 4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes. Life Sciences, 2006, 79: 1428-1435.

[14]Yonezawa T, Haga S, Kobayashi Y, Katoh K, Obara Y. Saturated fatty acids stimulate and insulin suppresses BMCP1 expression in bovine mammary epithelial cells. Biochemical and Biophysical Research Communications, 2009, 390: 915-919.

[15]Ho P W, Ho J W, Liu H F, So D H, Tse Z H, Chan K H, Ramsden D B, Ho S L. Mitochondrial neuronal uncoupling proteins: a target for potential disease-modification in Parkinson's disease. Translational Neurodegeneration, 2012, 1(1): 3.

[16]Gasior M, Rogawski M A, Hartman A L. Neuroprotective and disease-modifying effects of the ketogenic diet. Behavioural Pharmacology, 2006, 17: 431-439.

[17]袁亚男, 刘文忠, 刘建华, 乔利英, 武建亮. 绵羊 UCP1 基因碱基突变与其蛋白结构和功能. 中国农业科学, 2012, 45: 2973-2980.

Yuan Y N, Liu W Z, Liu J H, Qiao L Y, Wu J L. The base mutation of ovine UCP1 gene and its relationship with protein structure and function. Scientia Agricultura Sinica, 2012, 45: 2973-2980. (in Chinese)

[18]Rial E, Zardoya R. Oxidative stress, thermogenesis and evolution of uncoupling proteins. Journal of Biology, 2009, 8(6): 58.

[19]Krauss S, Zhang C Y, Lowell B B. The mitochondrial uncoupling protein homologues. Nature Reviews Molecular Cell Biology, 2005, 6: 248-261.

[20]Ledesma A, de Lacoba M G, Rial E. The mitochondrial uncoupling proteins. Genome Biology, 2002, 3(12): 3015.1-3015.9.

[21]von Heijne G. The signal peptide. The Journal of Membrane Biology, 1990, 115: 195-201.

[22]Ridley R G, Patel H V, Gerber G E, Morton R C, Freeman K B. Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence. Nucleic Acids Research, 1986, 14: 4025-4035.

[23]Neupert W, Herrmann J M. Translocation of proteins into mitochondria. Annual Review of Biochemistry, 2007, 76: 723-749.

[24]Moremen K W, Tiemeyer M, Nairn A V. Vertebrate protein glycosylation: diversity, synthesis and function. Nature Reviews Molecular Cell Biology, 2012, 13: 448-462.

[25]Manning C, Plowman G D, Hunter T, Sudarsanam S. Evolution of protein kinase signaling from yeast to man. Trends in Biochemical Sciences, 2002, 27: 514-520.

[26]Cie?la J, Fr?czyk T, Rode W. Phosphorylation of basic amino acid residues in proteins: important but easily missed. Acta Biochimica Polonica, 2011, 58: 137-148.

[27]Wei Z, Chigurupati S, Bagsiyao P, Henriquez A, Chan S L. The brain uncoupling protein UCP4 attenuates mitochondrial toxin induced cell death: role of extracellular signal-regulated kinases in bioenergetics adaptation and cell survival. Neurotoxicity Research, 2009, 16: 14-29.

[28]Cohen P. The regulation of protein function by multisite phosphorylation-A 25-year update. Trends in Biochemical Sciences, 2000, 25: 596-601.

[29]Hanak P, Jezek P. Mitochondrial uncoupling proteins and phylogenesis-UCP4 as the ancestral uncoupling protein. Federation of European Biochemical Societies Letters, 2001, 495: 137-141.

[30]Chan S L, Liu D, Kyriazis G A, Bagsiyao P, Ouyang X, Mattson M P. Mitochondrial uncoupling protein-4 regulates calcium homeostasis and sensitivity to store depletion induced apoptosis in neural cells. Journal of Biological Chemistry, 2006, 281: 37391-37403.

[31]周旭华, 郭锡熔, 金子林, 倪毓辉, 潘晓勤, 陈荣华. 解偶联蛋白4基因在3T3-L1脂肪细胞诱导分化中的表达水平. 实用儿科临床杂志, 2004, 19: 637-639.

Zhou X H, Guo X R, Jin Z L, Ni Y H, Pan X Q, Chen R H. Uncoupling protein 4 gene expression during 3T3-L1 preadipocyte differentiation. Journal of Applied Clinical Pediatrics, 2004, 19: 637-639. (in Chinese)

[32]Denjean F, Lachuer J, Géloën A, Cohen-Adad F, Moulin C, Barré H, Duchamp C. Differential regulation of uncoupling protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats. FEBS Letters, 1999, 444: 181-185.