牦牛CYGB基因CDS区克隆与生物信息学分析  

Cloning and Bioinformatics Analysis on CDS of CYGB Gene in Yak

作　　者：孙雪婧[1,2] 杜晓华[1,2] 杨孝朴[1] 罗玉柱[2] 刘霞[3,2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]甘肃农业大学动物科学技术学院/甘肃省草食动物生物技术重点实验室,兰州730070 [3]甘肃农业大学生命科学技术学院,兰州730070

出　　处：《中国农业科学》2014年第13期2690-2698,共9页Scientia Agricultura Sinica

基　　金：甘肃省教育厅研究生导师科研项目计划(1102-04);甘肃省高等学校基本科研业务费资助项目(2013)

摘　　要：【目的】丰富牦牛CYGB基因研究的基础数据,对牦牛CYGB基因的CDS区进行克隆和生物信息学分析。【方法】提取牦牛大脑海马区组织的总RNA并运用RT-PCR技术反转录为cDNA,并根据GenBank中普通牛CYGB基因cDNA序列(GenBank登录号:DV874786.1),使用Primer3.0在线软件设计特异性引物,运用PCR扩增技术、TA克隆技术和核酸测序技术获得CYGB基因的完整CDS区序列及部分5′端和3′端UTR区,并使用ProtParam、PredictProtein、SWISS-MODEL等在线分析软件与Lasergene7.1软件包分析CYGB的一级结构、二级结构、三级结构与理化性质,并进行同源性分析及构建系统进化树;利用PyMol软件修饰并输出三维结构;使用在线亚细胞定位工具PSORT II Prediction预测蛋白质的亚细胞定位;使用Protfun软件对蛋白质的功能进行预测分析。【结果】克隆获得牦牛CYGB基因650 bp,包括CDS区573 bp(GenBank登录号:KF669898),碱基组成为A 20.59%、T 16.40%、G 33.33%、C 29.67%,编码190个氨基酸残基组成的蛋白质。与普通牛比对,牦牛CYGB基因在CDS区存在4个碱基突变,同源性为99.3%,这个突变未导致氨基酸序列的改变,4个突变均属同义突变。牦牛CYGB基因编码蛋白的分子式为C964H1513N263O278S7,分子量约为21.5 kD,理论等电点(pI)为6.32,消光系数为24075,不稳定系数为48.43,疏水指数为83.63,平均亲水性为-0.301,属不稳定可溶性酸性蛋白质,在哺乳动物网织红细胞内的半衰期为30 h。二级结构以α-螺旋和无规卷曲为主,其中α-螺旋占64.21%,无规卷曲占35.79%,属全α类蛋白质。三级结构是一个呈"three-over-three"三明治夹心型的α-螺旋折叠结构。亚细胞定位CYGB分布在细胞质(65.2%)、细胞核(17.4%)、线粒体(13.0%)、分泌系统的囊泡(4.3%)中,主要在细胞质,推测可能在能量代谢和辅因子的生物合成过程中发挥信号转导和转录因子调控的作用。牦牛CYGB氨基酸序列与普通牛、绵羊、家犬、小�[Objective]In order to enrich basic data in yak CYGB gene, CDS region of yak CYGB gene was cloned and analyzed by bioinformatics method. [Method] Total RNA of yak hippocampus tissue was extracted and reverse transcribed into cDNA by RT-PCR technology. Specific primers were designed according to cDNA sequence of cattle CYGB gene in the GenBank（GenBank accession No.：DV874786.1） by online software Primer 3.0. The CDS region and part of 5′ UTR and 3′ UTR in yak CYGB gene were cloned from yak hippocampus total RNA by PCR amplification, TA cloning and nucleic acid sequencing technology. The primary structure, secondary structure, tertiary structure, physicochemical properties, homology were analyzed and phylogenetic tree of CYGB was constructed by online software like ProtParam, PredictProtein, SWISS-MODEL and Lasergene7.1 software package. The three-dimensional structure was modified and output by PyMol software. The protein subcellular localization was predicted by online subcellular localization tool PSORT II Prediction, and the protein function was predicted by Protfun software.[Result]The 650 bp length CYGB gene in yak was got by cloning, including the 573 bp length CDS region（GenBank accession No.：KF669898）, and the bases composition were 20.59%A, 16.40%T, 33.33%G, 29.67%C, encoding 190 amino acids. Alignment with CDS and amino acid sequence of cattle CYGB gene, four base mutations were found and amino acid was not mutated, four mutations are synonymous mutations. The formula of protein encoded by CYGB gene in yak was C964H1513N263O278S7, and the molecular weight was 21.5 kD, the theory isoelectric point was 6.32, the extinction coefficient was 24075, the instability index was 48.43, the aliphatic index was 83.63, and the grand average of hydropathicity was-0.301. It was an unstable and soluble protein. Its estimated half-life is 30 hours in mammal reticulocyte. The secondary structure of CYGB was mainly α-helices and random coil, α-helices was 64.21% and random coil was 35.79%, CYGB was an α c

关 键 词：牦牛 胞红蛋白 基因克隆 生物信息学 

分 类 号：S823.85[农业科学—畜牧学]