鲢神经肽Y基因的克隆及其在禁食-恢复投喂条件下的表达特征分析

杨镇宇; 王丹; 李忠; 梁宏伟; 赵金坤; 罗相忠; 邹桂伟

doi:10.7541/2016.33

鲢神经肽Y基因的克隆及其在禁食-恢复投喂条件下的表达特征分析

杨镇宇^1,2,
王丹^2,3,
李忠²,
梁宏伟²,
赵金坤^1,2,
罗相忠²,
邹桂伟^2, ,

1.
华中农业大学水产学院, 武汉 430070
2.
中国水产科学研究院长江水产研究所, 武汉 430223
3.
中国水产科学研究院淡水渔业研究中心, 无锡 214081

基金项目:

现代农业产业技术体系建设专项资金(CARS-46-01)

十二五科技支撑计划(2012BAD26B02)

中央级公益性科研院所基本科研业务费专项资金(2015JBFM39)

农业部热带亚热带水产资源利用与养殖重点实验室开放课题

中央高校基本科研业务费专项基金资助(2013SC14)资助

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出版历程
- 收稿日期: 2015-02-04
- 修回日期: 2015-05-19
- 发布日期: 2016-03-24

CLONING AND EXPRESSION OF NPY IN THE SILVER CARP (HYPOPHTHALMICHTHYS MOLITRIX) DURING FASTING AND REFEEDING

1.
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China;
2.
Yangtze River Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Wuhan 430223, China;
3.
Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences, Wuxi 214081, China

Funds:

Supported by the earmarked fund for China Agriculture Research System (No. CARS-46-01)

National Science and Technology Support Program (No. 2012BAD26B02)

National Nonprofit Institute Research Grant of Freshwater Fisheries Research Center (No. 2015JBFM39)

Key Laboratory of Tropical Subtropical Fishery Resource Application and Cultivation, Ministry of Agriculture

Fundamental Research Funds for the Central Universities of China (No. 2013SC14)

摘要

摘要: 为深入研究神经肽Y对鲢摄食活动的调节作用,研究利用同源克隆方法获得鲢NPY基因cDNA全长序列,并检测在禁食-恢复投喂条件下,NPY在脑和肝脏中的表达情况. 结果表明: 鲢NPY基因cDNA全长782 bp,包括5'端非翻译区68 bp,3'端非翻译区423 bp,开放阅读框291 bp,编码96个氨基酸; 氨基酸相似性比较和系统进化分析结果显示NPY较为保守; NPY在所检测的13个组织中均有表达,并且在脑垂体中表达量最大; 禁食导致NPY mRNA在脑中的表达量显著上升,恢复投喂6h后下降到基本水平,表明NPY对鲢摄食有促进作用; 在肝脏中,禁食前5天NPY mRNA表达量显著上升,禁食第7天急剧下降,恢复投喂4h后下降到基本水平; NPY mRNA在脑和肝脏中的表达具有组织差异性,其在肝脏中的具体作用机制有待进一步研究. 研究结果为探讨NPY在鲢中的生物学功能和在遗传育种中的作用提供了理论依据。
- 鲢 /
- NPY基因 /
- 禁食 /
- 恢复投喂 /
- 基因表达
Abstract: NPY plays a significant role in regulating food intake and energy metabolism in teleosts. To understand the biological role of NPY in the silver carp (Hypophthalmichthys molitrix), in this study we determined the full-length cDNA sequence of NPY and investigated the effect of fasting and refeeding on the level of NPY mRNA in the brain and the liver. The full cDNA sequence of NPY consisted of 782 bp, including a 291 bp open reading frame that potentially encodes 96 amino acids, a 68 bp 5'-untranslated region and a 423 bp 3'-untranslated region. Our study on the similarity of the amino acid sequence and the phylogenetic analysis of NPY demonstrated that the NPY gene was relatively conservative during the molecular evolution. The mRNA of NPY was ubiquitously expressed in all the tested tissues and especially abundant in the pituitary. In the brain, the expression of NPY mRNA increased significantly during fasting, and then decreased to the basal level at 6h after refeeding, which indicated that NPY could boost the food intake of the silver carp. In the liver, the expression of NPY mRNA increased significantly until 5d after fasting, then decreased sharply on the 7th day, and finally returned to the basal level at 4h after refeeding. NPY was expressed in a tissue-speci-fic pattern in both the brain and the liver. However, the function of NPY in the liver needs further study. Our study provided insights into the biological roles of NPY in the silver carp.
- Silver carp /
- NPY gene /
- Fasting /
- Refeeding /
- Gene expression analysis

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参考文献(41)

[1]	Matteri R L. Overview of central targets for appetite regulation [J]. Animal Science, 2001, 79: E148E158
[2]	Xie X J, Deng L, Zhang B, et al. Advances and studies on ecophysiological effects of starvation on fish [J]. Acta Hydrobiologica Sinica, 1998, 22(2): 181188 [谢小军, 邓利, 张波, 等. 饥饿对鱼类生理生态学影响的研究进展. 水生生物学报, 1998, 22(2): 181188]
[3]	Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide [J]. Nature, 1982, 296(5858): 659660
[4]	Stanley B G, Kyrkouli S E, Lampert S, et al. Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity [J]. Peptides, 1986, 7(6): 11891192
[5]	Woods S C, Figlewicz D P, Madden L, et al. NPY and food intake: discrepancies in the model [J]. Regulatory Peptides, 1998, (7576): 403408
[6]	Gehlert D R. Role of hypothalamic neuropeptide Y in feeding and obesity [J]. Neuropeptides, 1999, 33(5): 329338
[7]	Liao W Q, Liang X F, Fang L. Research of NPY gene and its appetite regulation mechanism [J]. Water Conservancy Related Fisheries, 2005, 25(4): 3537 [廖婉琴, 梁旭方, 方玲. 鱼类神经肽Y基因及其食欲调控机理研究. 水利渔业, 2005, 25(4): 3537]
[8]	Silverstein J T, Plisetskaya E M. The effects of NPY and insulin on food intake regulation in fish [J]. Integrative and Comparative Biology, 2000, 40(2): 296308
[9]	Wang Q, Tan X G, Sun W, et al. Expression of recombinant flounder NPY protein in vitro and detection of endogenous NPY in flounder [J]. Marine Sciences, 2014, 38(4): 1519 [王倩, 谭训刚, 孙威, 等. 牙鲆NPY的体外表达及体内检测. 海洋科学, 2014, 38(4): 1519]
[10]	Lpez-Patio M A, Guijarro A I, Isorna E, et al. Neuropeptide Y has a stimulatory action on feeding behavior in goldfish (Carassius auratus) [J]. European Journal of Pharmacology, 1999, 377(23): 147153
[11]	Carpio Y, Acosta J, Morales A, et al. Cloning, expression and growth promoting action of Red tilapia (Oreochromis sp.) neuropeptide Y [J]. Peptides, 2006, 27(4): 710718
[12]	Peterson B C, Waldbieser G C, Riley L G, et al. Pre-and postprandial changes in orexigenic and anorexigenic factors in channel catfish (Ictalurus punctatus) [J]. General and Comparative Endocrinology, 2012, 176(2): 231239
[13]	Zhang S M, Deng H, Wang D Q, et al. Population structure and genetic diversity of silver carp and grass carp from populations of Yangtze River system revealed by RAPD [J]. Acta Hydrobiologica Sinica, 2001, 25(4): 324330 [张四明, 邓怀, 汪登强, 等. 长江水系鲢和草鱼遗传结构及变异性的RAPD研究. 水生生物学报, 2001, 25(4): 324330]
[14]	China Fishery Statistical Yearbook [M]. Beijing: Chinese Agriculture Press. 20042014 [中国渔业统计年鉴. 北京: 中国农业出版社. 20042014]
[15]	Huising M O, Geven E J W, Kruiswijk C P, et al. Increased leptin expression in common carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation [J]. Endocrinology, 2006, 147(12): 57865797
[16]	Lindblom J, Haitina T, Fredriksson R, et al. Differential regulation of nuclear receptors, neuropeptides and peptide hormones in the hypothalamus and pituitary of food restricted rats [J]. Molecular Brain Research, 2005, 133(1): 3746
[17]	Tanic N, Perovic M, Mladenovic A, et al. Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus evaluation by real time RT-PCR [J]. Journal of Molecular Neuroscience, 2007, 32(1): 3846
[18]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-CT method [J]. Methods, 2001, 25(4): 402408
[19]	Mattews J E, Jansen M, Lyerly D, et al. Pharmacological characterization and selectivity of the NPY antagonist GR231118 (1229U91) for different NPY receptors [J]. Regulatory Peptides, 1997, 72(23): 113119
[20]	Yu Y, Liang X F, Li S Y, et al. Molecular cloning and evolutional analysis of NPY, UCP2, LPL and HL gene of Largemouth Bass (Micropterus salmoides) [J]. Acta Hydrobiologica Sinica, 2008, 32(6): 900907 [于燕, 梁旭方, 李诗盈, 等. 大口黑鲈脂代谢相关基因NPY, UCP2, LPL, HL克隆与分子进化分析. 水生生物学报, 2008, 32(6): 900907]
[21]	Aboumder R, Elhusseiny A, Cohen Z, et al. Expression of neuropeptide Y receptors mRNA and protein in human brain vessels and cerebromicrovascular cells in culture [J]. Journal of Cerebral Blood Flow and Metabolism, 1999, 19(2): 155163
[22]	Heilig M, Widerlv E. Neuropeptide Y: an overview of central distribution, functional aspects, and possible involvement in neuropsychiatric illnesses [J]. Acta Psychiatrica Scandinavica, 1990, 82(2): 95114
[23]	Doyon C, Gilmour K M, Trudeau V L, et al. Corticotropin-releasing factor and neuropeptide Y mRNA levels are elevated in the preoptic area of socially subordinate rainbow trout [J]. General and Comparative Endocrinology, 2003, 133(2): 260271
[24]	Chen R. Cloning, prokaryotic expression and functions of NPY in orange-spotted grouper (Epinephelus coioides) [D]. Thesis for master of Science. Sun Yat-Sen University, Guangzhou. 2006 [陈蓉. 斜带石斑鱼神经肽Y基因的克隆、原核表达与功能研究. 硕士学位论文, 中山大学, 广州. 2006]
[25]	Yang Y, Peng H W, Li Y L, et al. Molecular cloning and tissue expression analysis of the NPY gene in Silurus meridionalis [J]. Journal of Henan University (Natural Science), 2013, 43(3): 295300 [杨雁, 彭焕文, 李艳利, 等. 南方鲇 (Silurus meridionalis) 神经肽Y基因cDNA克隆及组织表达分析. 河南大学学报 (自然科学版), 2013, 43(3): 295300]
[26]	Murashita K, Kurokawa T, Ebbesson L O E, et al. Characterization, tissue distribution, and regulation of agouti-related protein (AgRP), cocaine-and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) in Atlantic salmon (Salmo salar) [J]. General and Comparative Endocrinology, 2009, 162(2): 160171
[27]	Cao L, Liang H W, Li Z, et al. Cloning and expression analysis of NPY gene in yellow catfish (Pelteobagrus fulvidraco) [J]. Journal of Northwest A and F University (Natural Science Edition), 2013, 41(7): 17 [曹磊, 梁宏伟, 李忠, 等. 黄颡鱼神经肽Y基因 (NPY) cDNA全序列的克隆及其表达特征分析. 西北农林科技大学学报 (自然科学版), 2013, 41(7): 17]
[28]	Kehoe A S, Volkoff H. Cloning and characterization of neuropeptide Y (NPY) and cocaine and amphetamine regulated transcript (CART) in Atlantic cod (Gadus morhua) [J]. Comparative Biochemistry and Physiology Part A: Molecular and Integrative Physiology, 2007, 146(3): 451461
[29]	Liu Z F. The progress of checking methods on pituitary disease [J]. Introduction of Japanese Medical Science, 1995, 16(2): 8889 [刘志凡. 垂体疾病检查方法的进展. 日本医学介绍, 1995, 16(2): 8889]
[30]	Macdonald E, Volkoff H. Cloning, distribution and effects of season and nutritional status on the expression of neuropeptide Y (NPY), cocaine and amphetamine regulated transcript (CART) and cholecystokinin (CCK) in winter flounder (Pseudopleuronectes americanus) [J]. Hormones and Behavior, 2009, 56(1): 5865
[31]	Macdonald E, Volkoff H. Neuropeptide Y (NPY), cocaine and amphetamine-regulated transcript (CART) and cholecystokinin (CCK) in winter skate (Raja ocellata): cDNA cloning, tissue distribution and mRNA expression responses to fasting [J]. General and Comparative Endocrinology, 2009, 161(2): 252261
[32]	Li Z H. The cDNA cloning and characterization analysis of NPY gene in Mystus macropterus Bleeker [D]. Thesis for master of Science. School of Life Science Southwest University, Chongqing. 2011 [李振华. 大鳍鳠NPY基因cDNA克隆及特征分析. 硕士学位论文, 西南大学生命科学学院, 重庆. 2011]
[33]	Cerd-Reverter J M, Martnez-Rodrguez G, Zanuy S, et al. Cloning the neuropeptide Y exon 2 from sea bass (Dicentrarchus labrax) [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1999, 123(2): 181186
[34]	Dumont Y, Martel J C, Fournier A, et al. Neuropeptide Y and neuropeptide Y receptor subtypes in brain and peripheral tissues [J]. Progress in Neurobiology, 1992, 38(2): 125167
[35]	Shahbazi F, Holmgren S, Larhammar D, et al. Neuropeptide Y effects on vasorelaxation and intestinal contraction in the Atlantic cod Gadus morhua [J]. American Journal of Physiology, 2002, 282(5): 14141421
[36]	Bjenning C, Hazon N, Balasubramaniam A, et al. Distribution and activity of dogfish NPY and Peptide YY in the cardiovascular system of the common dogfish [J]. American Journal of Physiology, 1993, 264(6): 11191124
[37]	Bjenning C, Holmgren S, Farrell A P. Neuropeptide Y potentiates contractile response to norepinephrine in skate coronary artery [J]. American Journal of Physiology, 1993, 265(2): 661665
[38]	Kohno D, Yada T. Arcuate NPY neurons sense and integrate peripheral metabolic signals to control feeding [J]. Neruopeptides, 2012, 46(6): 315319
[39]	Wang J, Wang Y J. Influence of different diet on NPY expression in arcuate nucleus of rats hypothalamus [J]. Journal of Chengde Medical College, 2012, 29(3): 241243 [王晶, 王英杰. 不同膳食对大鼠下丘脑弓状核NPY表达的影响. 承德医学院学报, 2012, 29(3): 241243]
[40]	Narnaware Y K, Peter R E. Effects of food deprivation and refeeding on neuropeptide Y (NPY) mRNA levels in goldfish [J]. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2001, 129(23): 633637
[41]	Sucajtys-Szulc E, Goykea E, Korczynska J, et al. Chronic food restriction differentially affects NPY mRNA level in neurons of the hypothalamus and in neurons that innervate liver [J]. Neuroscience Letters, 2008, 433(3): 174177