《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (9): 2358-2367.doi: 10.16431/j.cnki.1671-7236.2018.09.002

• 生物技术 • 上一篇    下一篇

奶水牛固醇携带蛋白2基因克隆、生物信息学分析及其组织表达检测

梁莎莎, 庞春英, 邓廷贤, 马小娅, 陆杏蓉, 段安琴, 梁贤威   

  1. 中国农业科学院广西水牛研究所, 农业部(广西)水牛遗传繁育重点实验室, 南宁 530001
  • 收稿日期:2018-03-09 出版日期:2018-09-20 发布日期:2018-09-26
  • 通讯作者: 梁贤威 E-mail:liangbri@126.com
  • 作者简介:梁莎莎(1992-),女,广西南宁人,学士,研究实习员,研究方向:水牛分子遗传学,E-mail:396066793@qq.com
  • 基金资助:

    科技部国际科技合作项目(2014DFA31970);广西科技重大专项桂科(AA16450002);水牛基(1705005);广西水产畜牧科技推广应用(桂渔牧科201633009)

Cloning, Bioinformatics and Tissue Expression Analysis of SCP2 Gene in Dairy Buffalo

LIANG Shasha, PANG Chunying, DENG Tingxian, MA Xiaoya, LU Xingrong, DUAN Anqin, LIANG Xianwei   

  1. Guangxi Key Laboratory of Buffalo Genetics, Breeding and Reproduction, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China
  • Received:2018-03-09 Online:2018-09-20 Published:2018-09-26

摘要:

本研究旨在对水牛固醇携带蛋白2(sterol carrier protein 2,SCP2)基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因(登录号:NM_001033990.3)为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632 bp,可编码543个氨基酸;其与黄牛、绵羊、山羊、白鲸、人、家犬和家猫的同源性分别为95.9%、93.4%、92.4%、89.4%、88.3%、86.3%和86.9%,说明SCP2基因CDS区在不同物种间具有较高的保守性。聚类分析则表明水牛与黄牛的分子进化关系最近;氨基酸序列分析表明,SCP2蛋白的分子式为C2602H4131N709O774S298,分子质量为58.66 ku,理论等电点(pI)为8.59,不稳定系数为27.94,平均亲水性为-0.215,属于碱性、稳定、亲水蛋白质;二级结构分析表明水牛SCP2蛋白由α-螺旋、无规则卷曲和延伸链构成,其中α-螺旋占35.54%,无规则卷曲占48.99%,延伸链占15.47%,与三级结构预测结果一致;亚细胞定位分析表明,水牛SCP2蛋白分布在细胞质(43.5%)、过氧化物酶体(21.7%)、线粒体(17.4%)、细胞核(13.0%)和细胞骨架(4.4%);跨膜结构和信号肽预测分析表明,水牛SCP2蛋白不含跨膜结构和信号肽;磷酸化位点分析发现,水牛SCP2蛋白有13个Ser、3个Thr和2个Tyr可能成为蛋白激酶磷酸化位点;蛋白质结合位点预测结果显示,水牛SCP2蛋白含有12个蛋白质结合位点和1个多核苷酸结合位点;实时荧光定量PCR结果表明,水牛SCP2基因在肝脏中表达量最高,其他组织中表达量从高到低依次为乳腺、淋巴、肾脏、大肠、胃、肺脏、脾脏、卵巢、垂体、大脑和心脏。本试验为今后进一步探讨SCP2基因的功能奠定了基础。

关键词: 水牛; 固醇携带蛋白2(SCP2); 基因克隆; 生物信息学分析; 组织表达

Abstract:

The study was aimed to clone sterol carrier protein 2 (SCP2) of buffalo,analyze the sequences by bioinformatics software,and measure the expression of SCP2 gene in different tissues of buffalo.The complete CDS of buffalo SCP2 gene was successfully cloned which was 1 632 bp in length and encoded 543 amino acids using the SCP2 gene (GenBank accession No.:NM_001033990.3) of Bos taurus as the seed sequence.The CDS of buffalo SCP2 gene showed 95.9%,93.4%,92.4%,89.4%,88.3%,86.3% and 86.9% identity with that of Bos taurus,Ovis aries,Capra hircus,Delphinapterus leucas,Homo sapiens,Felis catus familiaris and Canis lupus,and the results of phylogenetic tree showed that the nearest relationship existed between buffalo and Bos taurus.The results of amino acid sequence analysis showed that the formula of protein encoded by SCP2 gene in buffalo was C2602H4131N709O774S298, and the molecular weight,theory isoelectric point,instability index and average of hydropathicity was 58.66 ku,8.59,27.94 and -0.215 respectively,showing it was an alkaline,hydrophilic and stable protein.The secondary structure of SCP2 protein mainly consisted of α-helices (35.54%),random coil (48.99%) and extended strand (15.47%),which was consistent with the results of tertiary structure prediction.The results of subcellular localization showed that SCP2 protein was located in the cytoplasm (43.5%),peroxisome (21.7%),mitochondria (17.4%),nuclear (13.0%),and cytoskeleton (4.4%).There was no transmembrane domain and signal peptide,indicating it was an non secretory protein.There were 13 Ser,3 Thr and 2 Tyr which might be a protein kinase phosphorylation site.Prediction of protein binding sites showed that buffalo SCP2 protein contained 12 protein binding sites and 1 polynucleotide binding site.The Real-time PCR results showed that the expression of buffalo SCP2 gene in the liver was obviously higher than other tissues,which followed by mammary gland,lymph,kidney,large intestine,stomach,lung,spleen,ovary,pituitary,brain and heart ranged from high to low.This study provided the basis for further studies on the function of SCP2 gene.

Key words: buffalo; sterol carrier protein 2(SCP2); gene cloning; bioinformatics analysis; tissue expression

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