鹌鹑VIPR-1的克隆、序列特征和组织表达分析

【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。

cDNA Cloning,Sequence Analysis and Tissue Specific Expression of Vasoactive Intestinal Peptide Type 1 Receptor(VIPR-1) in Quails

【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.