水牛SFRP 1基因序列分析、真核表达载体构建及组织表达分析  

Sequence Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of SFRP 1 Gene in Buffalo

作　　者：黄丽清 段安琴 郑海英 杨春艳 尚江华 HUANG Liqing;DUAN Anqin;ZHENG Haiying;YANG Chunyan;SHANG Jianghua(Guangxi Key Laboratory of Buffalo Genetics,Breeding and Reproduction,Guangxi Buffalo Research Institute,Nanning 530001,China;Key Laboratory of Buffalo Genetics,Breeding and Reproduction Technology,Ministry of Agriculture and Rural Affairs,Nanning 530001,China)

机构地区：[1]广西水牛研究所,广西水牛遗传繁育重点实验室,南宁530001 [2]农业农村部水牛遗传繁育技术重点实验室,南宁530001

出　　处：《中国畜牧兽医》2024年第5期1807-1818,共12页China Animal Husbandry & Veterinary Medicine

基　　金：广西科技重大专项(桂科AA22068099-1);广西自然科学基金(2021GXNSFAA196021);国家现代农业产业技术体系广西奶水牛产业创新团队(nycytxgxcxtd-2021-21-01)。

摘　　要：【目的】获取水牛分泌型卷曲相关蛋白1(SFRP1)基因CDS区序列,并预测其编码蛋白的结构功能,构建SFRP 1基因真核表达载体,检测SFRP 1基因在水牛不同组织中的表达情况,为探索SFRP 1基因在水牛生长发育中的作用奠定基础。【方法】以水牛卵巢组织cDNA为模板,通过RT-PCR对SFRP 1基因CDS区序列进行扩增并测序,利用生物信息学在线分析软件对水牛SFRP 1基因与不同物种进行比对和系统进化树构建,并预测SFRP1蛋白理化性质、信号肽、跨膜结构等。将获得的目的基因连接至pCMV-HAhyPBase-mcheery载体并转染至水牛颗粒细胞,检测转染后荧光和基因表达情况。通过实时荧光定量PCR检测水牛不同组织中SFRP 1基因表达情况。【结果】水牛SFRP 1基因CDS区长927 bp,共编码308个氨基酸。多重序列比对结果显示,水牛SFRP 1基因氨基酸序列与黄牛、牦牛、山羊、虎鲸、黑猩猩、北极狐、猫、人、小鼠的相似性分别为100%、100%、99.65%、98.58%、98.23%、98.23%、98.58%、98.23%、96.45%,存在CRD_FZ、NTR_like和DUF3367结构域。水牛SFRP 1基因核苷酸序列与黄牛、绵羊、山羊、牦牛、野牛、马鹿、野骆驼、猪、人的相似性分别为97.3%、95.9%、95.8%、94.5%、94.2%、93.7%、80.4%、78.5%和77.3%。系统进化树结果显示,水牛与黄牛、牦牛、野牛聚为一支。生物信息学分析结果显示,SFRP1蛋白呈碱性,为不稳定蛋白;第1—15位氨基酸处存在信号肽,为分泌型蛋白,存在跨膜结构,主要定位于细胞外;存在21个磷酸化位点和8个O-糖基化修饰位点。二级结构主要由α-螺旋、延伸链和无规则卷曲构成;水牛、黄牛、人的SFRP1蛋白三级结构高度相似。成功构建pCMV-mcheery-SFRP1真核表达载体并转染水牛颗粒细胞,转染72 h后pCMV-mcheery-SFRP1重组质粒组细胞内SFRP 1基因表达量极显著高于对照组(P<0.01)。实时荧光定量PCR结果显示,SFRP 1基因在水牛不同组织中�【Objective】The aim of this study was to obtain the CDS region sequence of the secreted fizzled-related protein 1(SFRP1)gene in buffalo,predict the structure and function of the encoded protein,construct the eukaryotic expression vector of SFRP 1 gene,and detect the expression of SFRP 1 gene in different tissues of buffalo,laying a foundation for exploring the role of SFRP 1 gene in the growth and development of buffalo.【Method】Using the cDNA of ovarian tissue as template,the CDS region sequence of SFRP 1 gene was amplified by RT-PCR and sequenced,and the SFRP 1 gene of buffalo was compared with different species by bioinformatics online analysis softwares,the phylogenetic tree was constructed,and the physicochemical properties,signal peptide,and transmembrane structure of SFRP1 protein were predicted.The obtained target gene was connected to pCMV-HAhyPBase-mcheery vector and transfected into buffalo granulosa cells.The fluorescence and gene expression were detected after transfection.The expression of SFRP 1 gene in different tissues of buffalo was detected by Real-time quantitative PCR.【Result】The CDS region of SFRP 1 gene in buffalo was 927 bp,encoding 308 amino acids.Multiple sequence comparison results showed that the amino acid sequence of SFRP 1 gene similarity between buffalo and Bos taurus,Bos mutus,Capra hircus,Orcinus orca,Pan troglodytes,Vulpes lagopus,Felis catuswas,Homo sapiens and Mus musculus were 100%,100%,99.65%,98.58%,98.23%,98.23%,98.58%,98.23%and 96.45%,respectively.There were CRD_FZ,NTR_like and DUF3367 domains.The nucleotide sequence of SFRP 1 gene similarity between buffalo and Bos taurus,Ovis aries,Capra hircus,Bos mutus,Bison bison bison,Cervus elaphus,Camelus ferus,Sus scrofa and Homo sapiens were 97.3%,95.9%,95.8%,94.5%,94.2%,93.7%,80.4%,78.5%and 77.3%,respectively.Phylogenetic tree analysis showed that buffalo,Bos taurus,Bos mutus,and Bison bison bison converged into one group.Bioinformatics analysis showed that SFRP1 protein was alkaline and unstable.Signal peptide existed

关 键 词：水牛 SFRP 1基因 序列分析 真核表达载体 组织表达 

分 类 号：S823.83[农业科学—畜牧学]