鹌鹑VIPR-1的克隆、序列特征和组织表达分析

doi:10.3864/j.issn.0578-1752.2012.03.015

摘要/Abstract

摘要： 【目的】对鹌鹑血管活性肠肽1型受体（vasoacitve intestinal peptide type 1 receptor，VIPR-1）cDNA全长基因进行克隆及分析，为鹌鹑的分子育种提供基础资料。【方法】通过比较基因组学，采用RT-PCR和RACE技术，获得鹌鹑了VIPR-1 cDNA全长序列；通过生物学软件对其核苷酸序列和氨基酸序列进行了比对；采用实时定量PCR方法检测了VIPR-1在8个组织中的表达。【结果】鹌鹑VIPR-1的cDNA全长2 427 bp，包含了1 341 bp的开放性阅读框，编码446个氨基酸；序列分析显示，克隆获得的鹌鹑VIPR-1编码区序列与鸡该编码区序列存在41个碱基的差异，造成4个氨基酸残基的不同；VIPR-1氨基酸序列与鸡、火鸡、斑胸草雀的氨基酸的一致性分别为99.1%、92.2%、88%，与其它物种的一致性在60%—78%；各物种VIPR-1蛋白进化树符合物种进化规律；VIPR-1理化性质表明该蛋白为一偏碱性蛋白，蛋白二级结构主要由α-螺旋、β-折叠和β-转角构成；在N-端存在一个由22个氨基酸残基（MKSARLRVLLPLLGCLLSAASS）组成的信号肽，7个α-螺旋构成的跨膜域和C-端结构域，在跨膜域有胆固醇结合位点；在所检测的8个组织中VIPR-1 mRNA均有表达，在小肠中表达量最高。【结论】成功地克隆了鹌鹑VIPR-1 cDNA全长序列，该基因在小肠组织的表达高于其它组织，在跨膜域存在胆固醇结合位点。

关键词: 鹌鹑, 血管活性肠肽1型受体基因, 分子特征, 组织表达分析

Abstract: 【Objective】Vasoactive intestinal peptide type 1 receptor (VIPR-1) plays an important role in poultry reproduction, but the gene and its analysis have not been reported in quail prior to this study. The objective of this study was to clone the cDNA of quail VIPR-1, analyze its sequence and tissue expression pattern. 【Method】 Based on comparative genomics, the cDNA sequence of quail VIPR-1 was obtained by RT-PCR and RACE PCR. The nucleotide and amino acid sequences were analyzed and compared with that of other species. The expression levels of VIPR-1 in eight quail tissues were detected by real-time quantitative PCR. 【Result】 A 2 427 bp cDNA of the quail VIPR-1 was cloned and the length of its coding region was 1 341 bp, encoding a receptor precursor of 446 amino acids. There are 41 nucleotide differences in the coding region of VIPR-1 between quail and chicken, causing 4 amino acid differences in VIPR-1 protein. The quail VIPR-1 shows high amino acid sequence identity to that of chicken (99.1%), turkey (92.2%), and zebra finch (88.0%). Meanwhile, quail VIPR-1 also shares 60%-78% amino acid sequence identity to that of other species. The phylogenetic tree of VIPR-1 was consistent with the evolutionary relationship among species. The physical and chemical properties of quail VIPR-1 indicated that VIPR-1 was a basic protein. The secondary structure of quail VIPR-1 was made of α-helixes, β-sheets and β-turns. This protein contains a putative signal peptide (MKSARLRVLLPLLGCLLSAASS) in its N-terminus, 7 transmembrane domains, and a C-terminal hydrophilic domain. Real-time quantitative PCR assay demonstrated that the VIPR-1 mRNA could be detected in all 8 quail tissues examined with the highest expression level being noted in small intestinal.【Conclusion】The full length cDNA of quail VIPR-1 was cloned and the highest expression level of VIPR-1 was detected in small intestinal. In addition, the cholesyerol binding sites were identified on transmembrane domains of quail VIPR-1.

Key words: quail, VIPR-1, molecular characteristics, tissue expression

周敏, 李莹, 沈栩, 徐海平, 张成广, 张细权. 鹌鹑VIPR-1的克隆、序列特征和组织表达分析[J]. 中国农业科学, 2012, 45(3): 529-539.

ZHOU Min, LI Ying, SHEN Xu, XU Hai-Ping, ZHANG Cheng-Guang, ZHANG Xi-Quan. cDNA Cloning, Sequence Analysis and Tissue Specific Expression of Vasoactive Intestinal Peptide Type 1 Receptor (VIPR-1) in Quails[J]. Scientia Agricultura Sinica, 2012, 45(3): 529-539.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.03.015

https://www.chinaagrisci.com/CN/Y2012/V45/I3/529

参考文献

[1]Sharp P J. Genes for persistency of egg laying: White Leghorns and broodiness. Roslin Institute Edinburgh: Annual Report, 2004, 38-42.

[2]Liang Y, Cui J X, Yang G F, Leung F C, Zhang X Q. Polymorphisms of 5’ flanking region of chicken prolactin gene. Domestic Animal Endocrinology, 2006, 30(1): 1-16.

[3]Zhou M, Lei M M, Rao Y S, Nie Q H, Zeng H, Xia M N, Liang F F, Zhang D X, Zhang X Q. Polymorphisms of vasoactive intestinal peptide receptor-1 gene and their genetic effects on broodiness in chickens. Poultry Science, 2008, 87(5): 893-903.

[4]周敏, 梁菲菲, 饶友生, 曾华, 张德祥, 张细权. VIPR-1基因12个多态位点与鸡早期产蛋性状的相关性. 畜牧兽医学报, 2008, 39(9): 1147-1152.

Zhou M, Liang F F, Rao Y S, Zeng H, Zhang D X, Zhang X Q. Association of twelve polymorphisms of VIPR-1 gene with chicken early egg production traits. Acta Veterinaria et Zootechnica Sinica, 2008, 39(9): 1147-1152. (in Chinese)

[5]Vaudry D, Gonzalez B J, Basille M, Yon L, Fournier A, Vaudry H. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to function. Pharmacological Reviews, 2000, 52(2): 269-324.

[6]Perez F R, Camina J P, Menendez C, Beiras A, Casabiell X, Casanueva F. Cis-unsaturated free fatty acids block VIP-mediated GH and PRL secretion by perturbing the cAMP/protein kinase A pathway. Pituitary, 1998, 1(1): 25-32.

[7]Harmar A J. Family-B G-protein-coupled receptors. Genome Biology, 2001, 2(12): 3013.1-3013.10.

[8]Wang Y J, Li J, Wang C Y, Kwok A Y, Zhang X Q, Leung F C. Characterization of the receptors for chicken GHRH and GHRH-related peptides: Identification of a novel receptor for GHRH and the receptor for GHRH-LP (PRP). Domestic Animal Endocrinology, 2010, 38(1): 13-31.

[9]Kansaku N, Shimada K, Ohkubo T, Saito N, Suzuki T, Matsuda Y, Zadworny D. Molecular cloning of chicken vasoactive intestinal polypeptide receptor complementary DNA, tissue distribution and chromosomal localization. Biology of Reproduction, 2001, 64(5): 1575-1581.

[10]You S, Hsu C C, Kim H, Kho Y, Choi Y J, El Halawani M E, Farris J, Foster D N. Molecular cloning and expression analysis of the turkey vasoactive intestinal peptide receptor. General and Comparative Endocrinology, 2001, 124(1): 53-65.

[11]Cardoso J C, Vieira F A, Gomes A S, Power D M. PACAP, VIP and their receptors in the metazoan: insights about the origin and evolution of the ligand-receptor pair. Peptides, 2007, 28(9): 1902-1919.

[12]Laburthe M, Couvineau A. Molecular pharmacology and structure of VPAC receptors for VIP and PACAP. Regulatory Peptides, 2002, 108(2/3): 165-173.

[13]Tan Y V T, Couvineau A, Murail S, Ceraudo E, Neumann J M, Lacapere J J, Laburthe M. Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. The Journal of Biology Chemistry, 2006, 281(18): 12792-12798.

[14]Couvineau A, Fabre C, Gaudin P, Maoret J J, Laburthe M. Mutagenesis of N-glycosylation sites in the human vasoactive intestinal peptide 1 receptor. evidence that asparagine 58 or 69 is crucial for correct delivery of the receptor to plasma membrane. Biochemistry, 1996, 35(6): 1745-1752.

[15]Dickson L, Finlayson K. VPAC and PAC receptors: from ligands to function. Pharmacology and Therapeutics, 2009, 121(3): 294-316.

[16]Du K, Nicole P, Couvineau A, Laburthe M. Aspartate 196 in the first extracellular loop of the human VIP1 receptor is essential for VIP binding and VIP-stimulated cAMP production. Biochemical and Biophysical Research Communications, 1997, 230(2): 289-292.

[17]Couvineau A, Lacapere J J, Tan Y V, Rouyer-Fessard C, Nicole P, Laburthe M. Identification of cytoplasmic domains of hVPAC1 receptor required for activation of adenylyl cyclase. The Journal of Biological Chemistry, 2003, 278(27): 24759-24766.

[18]Lyu R M, Germano P M, Choi J K, Le S V, Pisegna J R. Identification of an eaaential amino acid motif within the C terminus of the pituitary adenylate cyclase-activating polypeptide type I receptor that is critical for signal transduction but not for receptor internalization. The Journal of Biological Chemistry, 2000, 275(46): 36134-36142.

[19]Chaiseha Y, Youngren O M, El Halawani M E. Expression of vasoactive intestinal peptide receptor messenger RNA in the hypothalamus and pituitary throughout the turkey reproductive cycle. Biology of Reproduction, 2004, 70(3): 593-599.

[20]El Halawani M E, Silsby J L, Rozenboim I, Pitts G R. Increased egg production by active immunization against vasoactive intestinal peptide in the turkey (Meleagris gallopavo). Biology of Reproduction, 1995, 52(1): 179-183.

[21]El Halawani M E, Whiting S E, Silsby J L, Pitts G R, Chaiseha Y. Active immunization with vasoactive intestinal peptide in turkey hens. Poultry Science, 2000, 79(3): 349-354.

[22]Chow B K, Yuen T T, Chan K W. Molecular evolution of vertebrate VIP receptors and functional characterization of a VIP receptor from goldfish carassius auratus. General and Comparative Endocrinology, 1997, 105(2): 176-185.

[23]Laburthe M, Couvinea A, Tan V. Class II G protein-coupled receptor for VIP and PACAP: structure, models of activation and pharmacology. Peptides, 2007, 28(9): 1631-1639.

[24]Hackett S J, Kimball R T, Reddy S, Bowie R C, Braun E L, Braun M J, Chojnowski J L, Cox W A, Han K L, Harshman J, Huddleston C J, Marks B D, Miglia K J, Moore W S, Sheldon F H, Steadman D W, Witt C C, Yuri T. A phylogenomic study of birds reveals their evolutionary history. Science, 2008, 320(5884): 1763-1768.

[25]Choi S S, Lahn B T. Adaptive evolution of MRG, a neuron-specific gene family implicated in nociception. Genome Research, 2003, 13(10): 2252-2259.

[26]Porter T E, Lopez M E, Mike R, Huberty A F. The increase in prolactin-secreting cells in incubating chicken hens can be mimicked by extended treatment of pituitary cells in vitro with vasoactive intestinal polypeptide(VIP). Domestic Animal Endocrinology, 2006, 30(2): 126-134.

[27]Pucadyil T J, Chattopadhyay A. Role of cholesterol in the function and organization of G-protein coupled receptors. Progress in Lipid Research, 2006, 45(4): 295-333.

[28]Paila Y D, Chattopadhyay A. Membrane cholesterol in the function and organization of G-protein coupled receptors. Subcell Biochemical, 2010, 51: 439-466.

[29]Rozenboin I, Pitts G R, Youngren O M, El Halawani M E. Effect of maternal passive immunization against vasoactive intestinal peptide on prolactin secretion in turkey poults. Biology of Reproduction, 1996, 54(3): 670-674.

[30]Tong Z, Pitts G R, You S, Foster D N, El Halawani M E. Vasoactive intestinal peptide stimulates turkey prolactin gene expression by increasing transcription rate and enhancing mRNA stability. Journal of Molecular Endocrinology, 1998, 21(3): 259-266.

[31]Kang S W, Gazzillo L C, You S, Wong E A, El Halawani M E. Turkey

prolactin gene regulation by VIP through 35-bp cis-acting element in the proximal promoter. General and Comparative Endocrinology, 2004, 138(2): 157-165.

[32]Kang S W, Youngren O M, El Halawani M E. Influence of VIP prolactinemia in turkey anterior pituitary cells: role of cAMP second messenger in VIP-induced prolactin gene expression. Regulatory Peptides, 2002, 109(1/3): 39-44.

[33]Dejda A, Matczak I, Wiktorowska-Owczarek A, Nowak J Z. Cyclic AMP formation in chicken brain: effect of vasoactive intestinal peptide, peptide histidine-isoleucine(PHI), and some PHI-related peptide. Polish Journal of Pharmacology, 2003, 55(5): 747-751.

[34]Zawilska J B, Niewiadomski P, Nowak J Z. Receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide in the goose cerebral cortex. Polish Journal of Pharmacology, 2004, 56(2): 203-211.

[35]Sun S, El Halawani M E. Protein kinase-C mediates chicken vasoactive intestinal peptide stimulated prolactin secretion and gene expression in turkey primary pituitary cells. General and Comparative Endocrinology, 1995, 99(3): 289-297.

[36]Nowak J Z, Dejda A, Zawilska J B. Pituitary adenylate cyclase- activating polypeptide (PACAP) and vasoactive intestinal peptide(VIP) activate protein kinase C in chick cerebral cortex. Polish Journal of Pharmacology, 2001, 53(6): 695-699.

[37]Said S I, Mutt V. Polypeptide with broad biological activity: isolation from small intestinal. Science, 1970, 169(3951): 1217-1218.

[38]Sherwood N M, Krueckl S L, McRory J E. The origin and function of the pituitary adenylate cyclase-activating polypeptide(PACAP)/ glucagon superfamily. Endocrine Reviews, 2000, 21(6): 619-670.

[39]Fahrenkrug J, Hannibal J. Neurotransmitters co-existing with VIP or PACAP. Peptides, 2004, 25(3): 393-401.