苏淮猪VRTN基因克隆、组织表达特征与多态性分析  

Sequence Cloning,Tissue Expression Profile and Polymorphism of VRTN Gene in Suhuai Pig

作　　者：吴一尘[1] 杜星[1] 李平华[1,2] 吴艳 王钧顺 刘红林[1] 李齐发[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095 [2]南京农业大学淮安研究院,江苏淮安223005 [3]淮阴种猪场,江苏淮安223322

出　　处：《中国农业科学》2016年第18期3639-3648,共10页Scientia Agricultura Sinica

基　　金：国家科技支撑计划(2015BAD03B01);江苏省农业科技自主创新资金(SCX(12)2097);南京农业大学SRT计划(1515A08)

摘　　要：【目的】获得苏淮猪VRTN基因编码区序列,了解其序列特征和组织表达特征,分析苏淮猪VRTN基因ins291位点的多态性。【方法】以苏淮猪卵巢组织c DNA为模板,采用克隆测序技术分离获得苏淮猪VRTN基因编码区序列。利用Bio Edit 7.0软件分析其序列特征和蛋白理化性质。利用Clustal W软件进行核苷酸和蛋白质序列比对分析。利用UCSC基因组浏览器与NCBI基因组数据库进行猪VRTN基因的染色体定位和基因组结构分析。利用SMART软件预测蛋白质功能域,CPHmodels软件预测蛋白质三级结构。随机选取3头成年苏淮猪母猪,屠宰后立即采集心、肝、脾、肺、肾、下丘脑、肌肉和卵巢等组织,提取组织总RNA,采用RT-PCR技术分析苏淮猪VRTN基因的组织表达谱。采集106头成年苏淮猪繁殖母猪耳组织样,提取基因组DNA,采用PCR-凝胶电泳分析苏淮猪VRTN基因ins291位点的多态性。【结果】苏淮猪VRTN基因编码区序列全长为2 097bp,与其它哺乳动物如人、小鼠、牛和羊的一致性分别为84.98%、74.53%、85.84%和86.45%,而与鸡的一致性只有54.42%。基因组结构分析发现猪VRTN基因由2个外显子和1个内含子构成,定位在猪7号染色体上。苏淮猪VRTN基因编码蛋白含有698个氨基酸残基,与人、小鼠、牛和羊等的一致性分别为85.55%、70.07%、86.20%和86.06%,但与鸡的一致性只有51.17%,可见哺乳动物VRTN基因在进化过程中比较保守。氨基酸组分分析显示苏淮猪VRTN蛋白氨基酸序列存在全部20种氨基酸,其中Leu(亮氨酸)含量最高,达到10.44%,而Asp天冬氨酸含量最低,只有1.43%。蛋白结构分析发现苏淮猪VRTN蛋白含有HTH结构域等典型结构域,由6个α螺旋、5个β折叠以及若干无规则卷曲组成。RT-PCR分析表明VRTN基因在苏淮猪心、肝、脾、肺、肾、下丘脑、卵巢和肌肉组织等组织中均有表达,说明VRTN基因是一个广泛表达的基因。在苏淮猪群体中检测[Objective] The aim of this study is to obtain the coding region sequence of Suhuai VRTN gene, identify its characteristics and expression patterns, analyze the polymorphism of VRTN ins291 in Suhuai pig population. [Method] The coding sequence of Suhuai pig VRTN gene was isolated by cloning and sequencing. The BioEdit 7.0 software was used to analyze the molecular characteristics and physicochemical properties of Suhuai pig VRTN. Nucleotide and amino acid sequences alignment was performed using Clustal W. Genomic organization and chromosomal locations were investigated by comparing the cDNA and corresponding genomic sequence （UCSC server and NCBI database）. Motif analysis was performed using the online programs SMART, and protein tertiary structure was predicted by CPHmodels server. Tissue samples of heart, liver, spleen, lung, kidney, hypothalamus, ovary and skeleton muscle were obtained from adult female Suhuai pigs （n=3）. Total RNA was extracted using a Trizol kit, and the expression patterns of VRTN gene of Suhuai pig were analyzed by RT-PCR. The ear samples were obtained from adult female Suhuai pigs （n=106）, and genomic DNA was extracted by a conventional phenol-chloroform extraction method. PCR-Gel electrophoresis was performed to analyze the polymorphism of VRTN ins291. [Result] The full length of the coding sequences of Suhuai pig VRTN gene is 2097 bp, which shared similarities of 84.98%, 74.53%, 85.84% and 86.45% to human, mouse, cattle and sheep, respectively, but only 54.42% to chicken. Genomic structure analysis showed that pig VRTN gene includes 2 exons and 1 intron, and is located on chromosome 7. The Suhuai pig VRTN gene encodes 698 amino acid residues, which shared similarities of 85.55%, 70.07%, 86.20% and 86.06% to human, mouse, cattle and sheep, respectively, but only 51.17% to chicken, indicated that VRTN is conserved in the evolution process of mammalian. Protein structure analysis found that VRTN protein of Suhuai pig contains several typical domains including HTH domain, and co

关 键 词：苏淮猪 VRTN基因 克隆 组织表达谱 多态性 

分 类 号：S828[农业科学—畜牧学]