Abstract：The uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier family, which play important roles in several physiological processes, including thermogenesis, reactive oxygen species generation, lipid metabolism and insulin secretion. Although the roles of UCP3 gene are well understood in mammals, little is known about it in pigeon (Columba livia). In this study, ten white king pigeons were selected as the research object. The full-length cDNA sequence of UCP3 gene was cloned from spleen by rapid amplification of cDNA ends (RACE) and analyzed using bioinformatics tools. The distribution and expression level of pigeon UCP3 in different tissues were determined by qRT-PCR. The results showed that the full-length of pigeon UCP3 (GenBank No. KU166860.1) cDNA was of 1 413 bp including a 924 bp open reading frame (ORF), 75 bp 5'UTR and 414 bp 3'UTR. The ORF encoded polypeptides of 307 amino acids. Structure prediction showed that there were three mitochondrial carrier motifs in the pigeon UCP3 protein. The deduced amino acid sequence of pigeon UCP3 shared identity of 90%~93% with other birds, 72%~75% with mammals, 70% with Danio rerio, and 69% with Xenopus laevis. The phylogenetic relationship analysis indicated that birds, mammals, fish and amphibians each formed a branch. And in the bird branch, Columba livia was closely related with Anas platyrhynchos. These results were similar to the biological classification of different species. The results of qRT-PCR showed that pigeon UCP3 were expressed in all 6 tissues examined. The UCP3 mRNA expression levels in lung were highest, which were significantly higher than that in other tissues (P＜0.05), followed by spleen and kidney, and relative lower expression was detected in muscle and heart, and the lowest expression was found in liver. The results of this work may provide a theoretical basis for the further study of its function in poultry.
[1]刘建华.2013. 绵羊UCP2和UCP3基因的克隆、表达及其遗传变异研究[D]. 博士学位论文, 山西农业大学, 导师: 刘文忠. pp. 95-113. (Liu J H. 2013. Study on the cloning, expression and genetic variation of ovine UCP2 and UCP3 genes[D]. Thesis for Ph.D., Shanxi Agricultural University, Supervisor: Liu W Z. pp. 95-113.) [2]彭广南.2013. 广西三黄鸡PPARγ和UCP3基因表达水平与脂肪性状的相关研究[D]. 硕士学位论文, 广西大学, 导师: 郭亚芬. pp. 32-55. (Peng G N. 2013. Corelation analysis between expression levels of PPARγ and UCP3 gene and meat quality traits in Guangxi sanhuang chicken[D]. Thesis for M.S., Guangxi University, Supervisor: Guo Y F. pp. 32-55.) [3]孙国权, 高树新, 吴慧光, 等.解偶联蛋白1、2 和3 基因在中国西门塔尔牛组织器官中的表达水平及其与胴体品质关系分析[J][J].华北农学报, 2014, 29(4):116-120 [4]徐红伟.2010. 兰州大尾羊脂肪代谢功能相关基因mRNA时空表达规律的研究[D]. 硕士学位论文, 西北民族大学, 导师: 臧荣鑫, 杨具田. pp. 63-68. (Xu H W. 2010. Temporal and spatial expression pattern of adipose metabolism related genes in Lanzhou fat-tailed sheep[D]. Thesis for M.S., Northwest Minzu University, Supervisor: Zang X R and Yang J T. pp. 63-68.) [5]张会丰, 高广亮, 王海威, 等.品种和饲养模式对鸡肉风味性状及其候选基因mRNA表达水平的影响[J][J].农业生物技术学报, 2014, 22(8):1018-1026 [6]Abe T, Mujahid A, Sato K, et al.Possible role of avian uncoupling protein in down-regulating mitochondrial superoxide production in skeletal muscle of fasted chickens[J][J].FEBS Letters, 2006, 580(20):4815-4822 [7]Bermejo-Nogales A, Calduch-Giner J A, Pérez-Sánchez J.Tissue-specific gene expression and functional regulation of uncoupling protein 2 (UCP2) by hypoxia and nutrient availability in gilthead sea bream (Sparus aurata): implications on the physiological significance of UCP1-3 variants[J][J].Fish Physiology and Biochemistry, 2014, 40(3):751-762 [8]Brand M D, Chien L F, Ainscow E K, et al.The causes and functions of mitochondrial proton leak[J][J].Biochimica et Biophysica Acta, 1994, 1187(2):132-139 [9]Busiello R A, Savarese S, Lombardi A.Mitochondrial uncoupling proteins and energy metabolism[J][J].Frontiers in Physiology, 2015, 6(/):36-/ [10]Carroll A M, Porter R K.Starvation-sensitive UCP3 protein expression in thymus and spleen mitochondria[J][J].Biochimica et Biophysica Acta, 2004, 1700(2):145-150 [11]Choi C S, Fillmore J J, Kim J K, et al.Overexpression of uncoupling protein 3 in skeletal muscle protects against fat- induced insulin resistance[J][J].The Journal of Clinical Investigation, 2007, 117(7):1995-2003 [12]Cioffi F, Senese R, de Lange P, et al.Uncoupling proteins: a complex journey to function discovery[J][J].BioFactors (Oxford, England), 2009, 35(5):417-428 [13]Clapham J C, Arch J R, Chapman H, et al.Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean[J][J].Nature, 2000, 406(6794):415-418 [14]Dos Santos R S, Galina A, Da-Silva W S.Cold acclimation increases mitochondrial oxidative capacity without inducing mitochondrial uncoupling in goldfish white skeletal muscle[J][J].Biology Open, 2013, 2(1):82-87 [15]Goglia F, Skulachev V P.A function for novel uncoupling proteins: antioxidant defense of mitochondrial matrix by translocating fatty acid peroxides from the inner to the outer membrane leaflet[J][J].FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 2003, 17(12):1585-1591 [16]Gracey A Y, Fraser E J, Li W, et al.Coping with cold: An integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate[J][J].Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(48):16970-16975 [17]Hesselink M K, Keizer H A, Borghouts L B, et al.Protein expression of UCP3 differs between human type 1, type 2a, and type 2b fibers[J][J].FASEB journal: official publication of the Federation of American Societies for Experimental Biology, 2001, 15(6):1071-1073 [18]Ishioka K, Kanehira K, Sasaki N, et al.Canine mitochondrial uncoupling proteins: structure and mRNA expression of three isoforms in adult beagles[J][J].Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 2002, 131(3):483-489 [19]Khalfallah Y, Fages S, Laville M, et al.Regulation of uncoupling protein-2 and uncoupling protein-3 mRNA expression during lipid infusion in human skeletal muscle and subcutaneous adipose tissue[J][J].Diabetes, 2000, 49(1):25-31 [20]Lee M S, Kim I H, Kim C T, et al.Reduction of body weight by dietary garlic is associated with an increase in uncoupling protein mRNA expression and activation of AMP-activated protein kinase in diet-induced obese mice[J].The Journal of Nutrition, 2011, 141(11):1947-1953 [21]Li H, Li Y, Zhao X, et al.Structure and nucleotide polymorphisms in pig uncoupling protein 2 and 3 genes[J][J].Animal Biotechnology, 2005, 16(2):209-220 [22]Lombardi A, Busiello R A, Napolitano L, et al.UCP3 translocates lipid hydroperoxide and mediates lipid hydroperoxide-dependent mitochondrial uncoupling[J][J].The Journal of Biological Chemisty, 2010, 285(22):16599-16605 [23]Mailloux R J, Harper M E.Uncoupling proteins and the control of mitochondrial reactive oxygen species production[J] [J].Free Radical Biology & Medicine, 2011, 51(6):1106-1115 [24]Matsuda J, Hosoda K, Itoh H, et al.Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet[J][J].FEBS Letters, 1997, 418(1-2):200-204 [25]Mensink M, Hesselink M K, Borghouts L B, et al.Skeletal muscle uncoupling protein-3 restores upon intervention in the prediabetic and diabetic state: implications for diabetes pathogenesis[J]?[J].Diabetes, Obesity & Metabolism, 2007, 9(4):594-596 [26]Mujahid A, Akiba Y, Toyomizu M.Olive oil-supplemented diet alleviates acute heat stress-induced mitochondrial ROS production in chicken skeletal muscle[J][J].American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 2009, 297(3):R690-698 [27]Oliveira B A, Pinhel M A, Nicoletti C F, et al.UCP1 and UCP3 expression is associated with lipid and carbohydrate oxidation and body composition[J][J].PLoS One, 2016, 11(3):e0150811-/ [28]Ozcan C, Palmeri M, Horvath T L, et al.Role of uncoupling protein 3 in ischemia-reperfusion injury, arrhythmias, and preconditioning[J][J].American Journal of Physiology. Heart and Circulatory Physiology, 2013, 304(9):H1192-1200 [29]Perrino C, Schiattarella G G, Sannino A, et al.Genetic deletion of uncoupling protein 3 exaggerates apoptotic cell death in the ischemic heart leading to heart failure[J][J].Journal of the American Heart Association, 2013, 2(3):e000086-/ [30]Ricquier D, Bouillaud F.Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance[J][J].The Journal of Physiology, 2000, 529(Pt 1):3-10 [31]Safari F, Anvari Z, Moshtaghioun S, et al.Differential expression of cardiac uncoupling proteins 2 and 3 in response to myocardial ischemia-reperfusion in rats[J][J].Life Sciences, 2014, 98(2):68-74 [32]Schrauwen P, Mensink M, Schaart G, et al.Reduced skeletal muscle uncoupling protein-3 content in prediabetic subjects and type 2 diabetic patients: restoration by rosiglitazone treatment[J]. , [J].The Journal of Clinical Endocrinology and Metabolism, 2006, 91(4):1520-1525 [33]Senese R, Valli V, Moreno M, et al.Uncoupling protein 3 expression levels influence insulin sensitivity, fatty acid oxidation, and related signaling pathways[J][J].Pflügers Archiv: European Journal of Physiology, 2011, 461(1):153-164 [34]Solanes G, Vidal-Puig A, Grujic D, et al.The human uncoupling protein-3 gene. Genomic structure, chromosomal localization, and genetic basis for short and long form transcripts[J][J].The Journal of Biological Chemistry, 1997, 272(41):25433-25436 [35]Son C, Hosoda K, Ishihara K, et al.Reduction of diet-induced obesity in transgenic mice overexpressing uncoupling protein 3 in skeletal muscle[J][J].Diabetologia, 2004, 47(1):47-54 [36]Song J W, Kim H J, Lee H, et al.Protective effect of peroxisome proliferator-activated receptor alpha activation against cardiac ischemia-reperfusion injury is related to upregulation of uncoupling protein-3[J][J].Oxidative Medicine and Cellular Longevity, 2016, 2016(/):3539649-/ [37]Toime L J, Brand M D.Uncoupling protein-3 lowers reactive oxygen species production in isolated mitochondria[J][J].Free Radical Biology & Medicine, 2010, 49(4):606-611 [38]Wen Z Y, Liang X F, He S, et al.Molecular cloning and tissue expression of uncoupling protein 1, 2 and 3 genes in Chinese perch (Siniperca chuatsi)[J]. , [J].Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 2015, 185(/):24-33 [39]Wu X，Gale P A.Small-molecule uncoupling protein mimics: synthetic anion receptors as fatty acid-activated proton transporters[J][J].Journal of the American Chemical Society, 2016, 138(50):16508-16514 [40]Yoshitomi H, Yamazaki K, Tanaka I.Cloning of mouse uncoupling protein 3 cDNA and 5'-flanking region, and its genetic map[J].Gene, 1998, 215(1):77-84
