团头鲂黑素皮质素3型受体基因克隆及表达分析
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摘要
黑素皮质素3型受体(MC3R)系统与动物摄食行为以及能量调控密切相关。为丰富鱼类MC3R相关基础研究,探索鱼类摄食行为与能量调控机制,本研究克隆了团头鲂(Megalobrama amblycephala)mc3r基因,采用分子生物信息学和相对荧光定量PCR方法分别对氨基酸序列保守结构域、组织表达分布和禁食条件下的表达变化等开展分析研究。结果显示,团头鲂mc3r基因编码区全长984 bp,编码327个氨基酸,与现有报道的MC3R氨基酸序列高度相似,具有典型的7次跨膜结构域。组织表达图谱分析表明,mc3rmRNA在下丘脑、垂体、肝脏和卵巢有相对较高的表达。禁食试验结果显示,下丘脑和垂体中的转录变化与周期性摄食信号相关,其中下丘脑mc3r mRNA在禁食72 h和14 d的表达显著上调(P<0.05),垂体mc3r mRNA在禁食72 h和禁食72 h后恢复投喂6 h表达量显著升高(P<0.05),而肝脏中mc3r mRNA在禁食48 h和14 d的表达量显著上调(P<0.05)。此外,对血液皮质醇和血糖的监测结果显示,两者均在禁食14 d有显著变化,其中皮质醇水平显著高于对照组(P<0.05),而血糖水平显著低于对照组(P<0.05)。综合本研究结果表明,MC3R在鲤科鱼类中具有高度相似的氨基酸序列和结构域;在组织中的转录表达变化与食物摄取有明显相关性,对调控鱼体摄食行为和能量代谢具有重要作用。
The melanocortin system consists of melanocortin peptides derived from the proopiomelanocortin gene,five melanocortin receptors, two endogenous antagonists, and two ancillary proteins. It plays a role in many important physiological functions of terrestrial animals. Studies on rodents have affirmed the role of melanocortin receptor 3(mc3 r) in feeding behavior and energy regulation. However, the function of mc3 r in fish, including basic information, is not well understood. Thus, to determine the initial value of fish mc3 r and assess the role of MC3 R in fish feeding behavior and energy regulation, the full-length cDNA of Megalobrama amblycephala mc3 r was cloned. The nucleotide sequence and deduced amino acid sequence were analyzed using bioinformatics methods. Blood glucose and cortisol levels were detected, and real-time quantitative PCR was employed to characterize the tissue expression profile and expression during fasting and refeeding. The results showed that M. amblycephala mc3 r cDNA encodes a protein of 327 amino acids, which shares a high amino acid sequence identity with that of other animals and is characteristic with the typical seven transmembrane domain. Phylogenetic analyses showed that the mc3 r cluster was within the MC3 R clade of the Cyprinidae MC3 Rs. The tissue expression analysis revealed that M. amblycephala mc3 r was highly expressed in the hypothalamus, pituitary, liver, and ovary,whereas its expression was much lower in the other detected tissues. During fasting and refeeding, mc3 r mRNA expression in the hypothalamus and pituitary showed a periodic variation with higher expression near the first feeding time every day both in the experimental and control groups. Furthermore, the expression of mc3 r mRNA was significantly increased and was higher than that in the control group after 72 h and 14 d fasting in the hypothalamus(P<0.05), whereas similar results occurred in the pituitary at 72 h fasting and 6 h after refeeding(P<0.05), and in the liver at 48 h and 14 d after fasting(P<0.05). In addition, an obvious change in blood glucose and cortisol was only observed with long term fasting(P<0.05). The M. amblycephala mc3 r is not only highly conservative in genetic information, but also in its tissue expression profile and physiological function during the evolutionary process, and may play an important role in feeding behavior and energy regulation.
The melanocortin system consists of melanocortin peptides derived from the proopiomelanocortin gene,five melanocortin receptors, two endogenous antagonists, and two ancillary proteins. It plays a role in many important physiological functions of terrestrial animals. Studies on rodents have affirmed the role of melanocortin receptor 3(mc3 r) in feeding behavior and energy regulation. However, the function of mc3 r in fish, including basic information, is not well understood. Thus, to determine the initial value of fish mc3 r and assess the role of MC3 R in fish feeding behavior and energy regulation, the full-length cDNA of Megalobrama amblycephala mc3 r was cloned. The nucleotide sequence and deduced amino acid sequence were analyzed using bioinformatics methods. Blood glucose and cortisol levels were detected, and real-time quantitative PCR was employed to characterize the tissue expression profile and expression during fasting and refeeding. The results showed that M. amblycephala mc3 r cDNA encodes a protein of 327 amino acids, which shares a high amino acid sequence identity with that of other animals and is characteristic with the typical seven transmembrane domain. Phylogenetic analyses showed that the mc3 r cluster was within the MC3 R clade of the Cyprinidae MC3 Rs. The tissue expression analysis revealed that M. amblycephala mc3 r was highly expressed in the hypothalamus, pituitary, liver, and ovary,whereas its expression was much lower in the other detected tissues. During fasting and refeeding, mc3 r mRNA expression in the hypothalamus and pituitary showed a periodic variation with higher expression near the first feeding time every day both in the experimental and control groups. Furthermore, the expression of mc3 r mRNA was significantly increased and was higher than that in the control group after 72 h and 14 d fasting in the hypothalamus(P<0.05), whereas similar results occurred in the pituitary at 72 h fasting and 6 h after refeeding(P<0.05), and in the liver at 48 h and 14 d after fasting(P<0.05). In addition, an obvious change in blood glucose and cortisol was only observed with long term fasting(P<0.05). The M. amblycephala mc3 r is not only highly conservative in genetic information, but also in its tissue expression profile and physiological function during the evolutionary process, and may play an important role in feeding behavior and energy regulation.
引文
[1]Gantz I,Fong T M.The melanocortin system[J].American Journal of Physiology Endocrinology and Metabolism,2003,284:468-474.
[2]Butler A A,Kesterson R A,Khong K,et al.A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse[J].Endocrinology,2000,141(9):3518-3521.
[3]Chen A S,Marsh D J,Trumbauer M E,et al.Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass[J].Nature Genetics,2000,26(1):97-102.
[4]Jiang S W,Peng J,Xiong Y Z.Melanocortin receptor involvement in the regulation of food intake and energy homeostasis[J].Hereditas,2002,24(2):223-226.[蒋思文,彭健,熊远著.黑素皮质素受体对动物采食量和能量稳态的调控[J].遗传,2002,24(2):223-226.]
[5]Rowland N E,Schaub J W,Robertson K L,et al.Effect of MTII on food intake and brain c-Fos in melanocortin-3,melanocortin-4,and double MC3 and MC4 receptor knockout mice[J].Peptides,2010,31(12):2314-2317.
[6]Pandit R,Omrani A,Luijendijk M C M,et al.Melanocortin3 receptor signaling in midbrain dopamine neurons increases the motivation for food reward[J].Neuropsychopharmacology,2016,41(9):2241-2251.
[7]Zhang Y B,Zeng R X,Du P,et al.Correlation analysis between MC3R and MC4R gene polymorphism and body weight in beagle dog[J].Chinese Journal of Animal Science,2012,48(13):13-16.[张轶博,曾瑞霞,杜鹏,等.比格犬MC3R和MC4R基因多态性与体重相关性的研究[J].中国畜牧杂志,2012,48(13):13-16.]
[8]Li S P,Du Z H,Ning F Y,et al.Correlation analysis between MC3R and MC4R gene polymorphism and growth traits in pigeon[J].Hereditas,2008,30(10):1333-1340.[李世鹏,杜智恒,宁方勇,等.鸽黑素皮质素受体3、4(MC3R、MC4R)基因多态性与生长性状的相关分析[J].遗传,2008,30(10):1333-1340.]
[9]Lee Y S,Poh L K S,Kek B L K,et al.The role of melanocortin 3 receptor gene in childhood obesity[J].Diabetes,2007,56(10):2622-2630.
[10]Timper K,Brüning J C.Hypothalamic circuits regulating appetite and energy homeostasis:pathways to obesity[J].Disease Models&Mechanisms,2017,10(6):679-689.
[11]Wikberg J E S,Mutulis F.Targeting melanocortin receptors:an approach to treat weight disorders and sexual dysfunction[J].Nature Reviews Drug Discovery,2008,7(4):307-323.
[12]Fen L,Xu Y,Li J L,et al.Molecular cloning and mutation analysis of MC3R gene in wild boar[J].Heilongjiang Animal Science and Veterinary Medicine,2009(6):18-20.[冯力,许尧,李金玲,等.野猪黑素皮质素受体3基因的克隆及变异初步研究[J].黑龙江畜牧兽医,2009(6):18-20.]
[13]Wang J,Xue M,Wu X F,et al.Regulation mechanism of selective feed intake of fish when fed different protein source diets:a review[J].Chinese Journal of Animal Nutrition,2014,26(4):833-842.[王嘉,薛敏,吴秀峰,等.鱼类对不同蛋白质源饲料选择性摄食调控机制的研究进展[J].动物营养学报,2014,26(4):833-842.]
[14]Klovins J,Haitina T,Ringholm A,et al.Cloning of two melanocortin(MC)receptors in spiny dogfish:MC3 receptor in cartilaginous fish shows high affinity to ACTH-derived peptides while it has lower preference to gamma-MSH[J].European Journal of Biochemistry,2004,271(21):4320-4331.
[15]Takahashi A,Davis P,Reinick C,et al.Characterization of melanocortin receptors from stingray Dasyatis akajei,a cartilaginous fish[J].General and Comparative Endocrinology,2016,232:115-124.
[16]Renquist B J,Zhang C,Williams S Y,et al.Development of an assay for high-throughput energy expenditure monitoring in the zebrafish[J].Zebrafish,2013,10(3):343-352.
[17]Yang J D,Wu S R,Pan S R,et al.Transcriptional level of melanocortin-3 receptor gene in goat[J].Journal of Southern Agriculture,2016,47(3):466-471.[杨家大,吴声榕,潘盛榕.山羊黑素皮质素受体-3基因转录水平差异分析[J].南方农业学报,2016,47(3):466-471.]
[18]Zhang D J,Liu D.Gene expression change of pig MC3R at cold stress[J].Journal of Animal Science and Veterinary Medicine,2012,31(1):20-21,23.[张冬杰,刘娣.冷应激下民猪黑素皮质素受体基因的表达变化[J].畜牧兽医杂志,2012,31(1):20-21,23.]
[19]Jiang S W,Jacobsson L,Kerje S,et al.Studies of relationship between the melanocortin-3 receptor gene and body weight in chicken for high and low weight lines`intercross[J].Acta Genetica Sinica,2002,29(4):322-325.[蒋思文,Jacobsson L,Kerje S,等.参考家系鸡黑素皮质素受体3基因多态性与体重关系研究[J].遗传学报,2002,29(4):322-325.]
[20]Xue C Y,Xi B W,Ren M C,et al.Molecular cloning,tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding[J].Chinese Journal of Oceanology and Limnology,2014,33(2):291-298.
[21]Gantz I,Konda Y,Tashiro T,et al.Molecular cloning of a novel melanocortin receptor[J].Journal of Biological Chemistry,1993,268:8246-8250.
[22]He X P,Zhou Y N,Yan Z X,et al.Molecular cloning and analysis and tissue distribution of melanocortin receptor 3and 4 genes in pigs(Sus scrofa)[J].Journal of Sichuan University(Natural Science edition),2013,50(4):899-907.[何夏萍,周彦妮,阎振鑫,等.猪黑皮质素受体3和4基因克隆及分析[J].四川大学学报(自然科学版),2013,50(4):899-907.]
[23]Takeuchi S,Takahashi S.A possible involvement of melanocortin 3 receptor in the regulation of adrenal gland function in the chicken[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,1999,1448(3):512-518.
[24]Sutton G M,Perez-Tilve D,Nogueiras R,et al.The melanocortin-3 receptor is required for entrainment to meal intake[J].Journal of Neuroscience,2008,28(48):12946-12955.
[25]Mavrikaki M,Girardet C,Kern A,et al.Melanocortin-3receptors in the limbic system mediate feeding-related motivational responses during weight loss[J].Molecular Metabolism,2016,5(7):566-579.
[26]Bertile F,Raclot T.The melanocortin system during fasting[J].Peptides,2006,27(2):291-300.
[2]Butler A A,Kesterson R A,Khong K,et al.A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse[J].Endocrinology,2000,141(9):3518-3521.
[3]Chen A S,Marsh D J,Trumbauer M E,et al.Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass[J].Nature Genetics,2000,26(1):97-102.
[4]Jiang S W,Peng J,Xiong Y Z.Melanocortin receptor involvement in the regulation of food intake and energy homeostasis[J].Hereditas,2002,24(2):223-226.[蒋思文,彭健,熊远著.黑素皮质素受体对动物采食量和能量稳态的调控[J].遗传,2002,24(2):223-226.]
[5]Rowland N E,Schaub J W,Robertson K L,et al.Effect of MTII on food intake and brain c-Fos in melanocortin-3,melanocortin-4,and double MC3 and MC4 receptor knockout mice[J].Peptides,2010,31(12):2314-2317.
[6]Pandit R,Omrani A,Luijendijk M C M,et al.Melanocortin3 receptor signaling in midbrain dopamine neurons increases the motivation for food reward[J].Neuropsychopharmacology,2016,41(9):2241-2251.
[7]Zhang Y B,Zeng R X,Du P,et al.Correlation analysis between MC3R and MC4R gene polymorphism and body weight in beagle dog[J].Chinese Journal of Animal Science,2012,48(13):13-16.[张轶博,曾瑞霞,杜鹏,等.比格犬MC3R和MC4R基因多态性与体重相关性的研究[J].中国畜牧杂志,2012,48(13):13-16.]
[8]Li S P,Du Z H,Ning F Y,et al.Correlation analysis between MC3R and MC4R gene polymorphism and growth traits in pigeon[J].Hereditas,2008,30(10):1333-1340.[李世鹏,杜智恒,宁方勇,等.鸽黑素皮质素受体3、4(MC3R、MC4R)基因多态性与生长性状的相关分析[J].遗传,2008,30(10):1333-1340.]
[9]Lee Y S,Poh L K S,Kek B L K,et al.The role of melanocortin 3 receptor gene in childhood obesity[J].Diabetes,2007,56(10):2622-2630.
[10]Timper K,Brüning J C.Hypothalamic circuits regulating appetite and energy homeostasis:pathways to obesity[J].Disease Models&Mechanisms,2017,10(6):679-689.
[11]Wikberg J E S,Mutulis F.Targeting melanocortin receptors:an approach to treat weight disorders and sexual dysfunction[J].Nature Reviews Drug Discovery,2008,7(4):307-323.
[12]Fen L,Xu Y,Li J L,et al.Molecular cloning and mutation analysis of MC3R gene in wild boar[J].Heilongjiang Animal Science and Veterinary Medicine,2009(6):18-20.[冯力,许尧,李金玲,等.野猪黑素皮质素受体3基因的克隆及变异初步研究[J].黑龙江畜牧兽医,2009(6):18-20.]
[13]Wang J,Xue M,Wu X F,et al.Regulation mechanism of selective feed intake of fish when fed different protein source diets:a review[J].Chinese Journal of Animal Nutrition,2014,26(4):833-842.[王嘉,薛敏,吴秀峰,等.鱼类对不同蛋白质源饲料选择性摄食调控机制的研究进展[J].动物营养学报,2014,26(4):833-842.]
[14]Klovins J,Haitina T,Ringholm A,et al.Cloning of two melanocortin(MC)receptors in spiny dogfish:MC3 receptor in cartilaginous fish shows high affinity to ACTH-derived peptides while it has lower preference to gamma-MSH[J].European Journal of Biochemistry,2004,271(21):4320-4331.
[15]Takahashi A,Davis P,Reinick C,et al.Characterization of melanocortin receptors from stingray Dasyatis akajei,a cartilaginous fish[J].General and Comparative Endocrinology,2016,232:115-124.
[16]Renquist B J,Zhang C,Williams S Y,et al.Development of an assay for high-throughput energy expenditure monitoring in the zebrafish[J].Zebrafish,2013,10(3):343-352.
[17]Yang J D,Wu S R,Pan S R,et al.Transcriptional level of melanocortin-3 receptor gene in goat[J].Journal of Southern Agriculture,2016,47(3):466-471.[杨家大,吴声榕,潘盛榕.山羊黑素皮质素受体-3基因转录水平差异分析[J].南方农业学报,2016,47(3):466-471.]
[18]Zhang D J,Liu D.Gene expression change of pig MC3R at cold stress[J].Journal of Animal Science and Veterinary Medicine,2012,31(1):20-21,23.[张冬杰,刘娣.冷应激下民猪黑素皮质素受体基因的表达变化[J].畜牧兽医杂志,2012,31(1):20-21,23.]
[19]Jiang S W,Jacobsson L,Kerje S,et al.Studies of relationship between the melanocortin-3 receptor gene and body weight in chicken for high and low weight lines`intercross[J].Acta Genetica Sinica,2002,29(4):322-325.[蒋思文,Jacobsson L,Kerje S,等.参考家系鸡黑素皮质素受体3基因多态性与体重关系研究[J].遗传学报,2002,29(4):322-325.]
[20]Xue C Y,Xi B W,Ren M C,et al.Molecular cloning,tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding[J].Chinese Journal of Oceanology and Limnology,2014,33(2):291-298.
[21]Gantz I,Konda Y,Tashiro T,et al.Molecular cloning of a novel melanocortin receptor[J].Journal of Biological Chemistry,1993,268:8246-8250.
[22]He X P,Zhou Y N,Yan Z X,et al.Molecular cloning and analysis and tissue distribution of melanocortin receptor 3and 4 genes in pigs(Sus scrofa)[J].Journal of Sichuan University(Natural Science edition),2013,50(4):899-907.[何夏萍,周彦妮,阎振鑫,等.猪黑皮质素受体3和4基因克隆及分析[J].四川大学学报(自然科学版),2013,50(4):899-907.]
[23]Takeuchi S,Takahashi S.A possible involvement of melanocortin 3 receptor in the regulation of adrenal gland function in the chicken[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,1999,1448(3):512-518.
[24]Sutton G M,Perez-Tilve D,Nogueiras R,et al.The melanocortin-3 receptor is required for entrainment to meal intake[J].Journal of Neuroscience,2008,28(48):12946-12955.
[25]Mavrikaki M,Girardet C,Kern A,et al.Melanocortin-3receptors in the limbic system mediate feeding-related motivational responses during weight loss[J].Molecular Metabolism,2016,5(7):566-579.
[26]Bertile F,Raclot T.The melanocortin system during fasting[J].Peptides,2006,27(2):291-300.