《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (12): 3317-3326.doi: 10.16431/j.cnki.1671-7236.2018.12.002

• 生物技术 • 上一篇    下一篇

GRP78基因克隆、生物信息学分析及组织表达谱研究

赵诗瑜, 张帆, 周晓龙, 汪涵, 赵阿勇, 杨松柏   

  1. 浙江农林大学动物科技学院, 临安 311300
  • 收稿日期:2018-04-13 出版日期:2018-12-20 发布日期:2018-12-21
  • 通讯作者: 杨松柏 E-mail:sbyang@zafu.edu.cn
  • 作者简介:赵诗瑜(1997-),女,山西长治人,本科,研究方向:动物遗传育种与繁殖,E-mail:zsy2463586863@163.com
  • 基金资助:

    国家自然科学基金(31501921);浙江农林大学学生科研训练项目(2013200005)

Cloning,Bioinformatics and Tissue Expression Analysis of Porcine GRP78 Gene

ZHAO Shiyu, ZHANG Fan, ZHOU Xiaolong, WANG Han, ZHAO Ayong, YANG Songbai   

  1. College of Animal Science and Technology, Zhejiang A & F University, Linan 311300, China
  • Received:2018-04-13 Online:2018-12-20 Published:2018-12-21

摘要:

本研究旨在克隆猪葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)基因,并进行生物信息学分析,探讨其在猪不同组织中的表达情况。根据GenBank上公布的猪GRP78基因序列(登录号:XM_001927795.6)设计引物,PCR扩增及测序获得猪GRP78基因CDS序列,使用在线软件分析GRP78的理化性质、跨膜结构、信号肽、疏水性、保守结构域、二级结构和三级结构,并进行同源性比对及系统进化树构建,利用实时荧光定量PCR方法检测猪GRP78基因在各组织中的表达情况。结果显示,猪GRP78基因CDS区长1 965 bp,可编码654个氨基酸。生物信息学分析表明,GRP78理论分子质量为72.3 ku,等电点(pI)为5.06,半衰期为30 h,其水溶液在280 nm处的消光系数为30 495,肽链N端为蛋氨酸(Met),不稳定系数为32.39,属于稳定蛋白。脂肪系数为85.40,总平均疏水指数为-0.496,无跨膜结构,存在信号肽序列,说明该蛋白属于分泌型蛋白;GRP78蛋白只包含1个超家族保守结构域:HSP70结构域。蛋白二级结构分析显示,GRP78蛋白中α-螺旋、延伸链、β-转角和无规则卷曲分别为40.06%、20.49%、8.10%和31.35%。同源性比对结果显示,猪GRP78基因与人(登录号:NM_005347.4)、小鼠(登录号:NM_001163434.1)、大鼠(登录号:NM_013083.2)、山羊(登录号:XM_005687138.3)、牛(登录号:NM_001075148.1)核苷酸序列的同源性分别为93%、91%、90%、95%、95%,氨基酸序列的同源性分别为99%、98%、98%、99%、99%,各个物种之间GRP78基因保守性较高。实时荧光定量PCR结果显示,GRP78基因在心脏、肝脏、脾脏、肺脏、肾脏、小肠、胃、卵巢、输卵管、乳腺、小脑、大脑、垂体等组织中均有表达,在心脏、脾脏、肺脏中相对高表达。本研究结果为今后深入研究GRP78基因的生物学功能提供了基础材料。

关键词: 猪; GRP78基因; 克隆; 生物信息学分析; 组织表达

Abstract:

This study was aimed to clone and analyze the CDS region of porcine glucose-regulated protein 78 (GRP78) gene using bioinformatics,and investigate the mRNA expression profile of GRP78 gene.Primers were designed according the predicted sequence of porcine GRP78 gene (accession No.:XM 001927795.6) in GenBank.The CDS region of porcine GRP78 gene was cloned by PCR amplification and sequencing.The physical and chemical property,transmembrane region,signal peptide,hydrophobicity,conserved domains,the secondary and tertiary structures were analyzed,and the phylogenetic tree of GRP78 gene was constructed by online prediction softwares.Finally,Real-time PCR was used to detect the tissue expression of porcine GRP78 gene.The results showed that the CDS of GRP78 gene was 1 965 bp,encoding 654 amino acids.Bioinformatics analysis showed that the molecular weight,theoretical electrical point (pI),half-life and the extinction coefficient of water solution at 280 nm of GRP78 were 72.3 ku,5.06,30 h,and 30 495,respectively.The peptide chain N-terminal of GRP78 was M (Met).The unstsble coefficient of GRP78 was 32.39,indicating that it belonged to the stable protein.The fat coefficient of GRP78 protein was 85.40,and the total average hydrophobic index was -0.496.GRP78 did not have transmembrane domain but it had one signal peptide,therefore,this protein belonged to the secreted protein.The secondary structure analysis of GRP78 showed that the percent of amino acids of α-helix,extended chain,β-turn and random coil were 40.06%,20.49%,8.10% and 31.35%,respectively.Homology analysis results showed that the nucleotide homology of GRP78 gene with human(NM_005347.4),mouse(NM_001163434.1),rat(NM_013083.2),goat (XM_005687138.3) and cattle (NM_001075148.1) were 93%,91%,90%,95% and 95%,whereas the amino acid homology were 99%,98%,98%,99% and 99%,respectively.These results indicated that GRP78 gene was highly conserved.Real-time PCR results showed that GRP78 gene was expressed in heart,liver,spleen,lung,kidney,small intestine,stomach,ovary,fallopian tube,breast,cerebellum,cerebrum and pituitary gland,and highly expressed in heart,spleen and lung.This study provided basic materials for further study of the biological function of GRP78 gene.

Key words: porcine; GRP78 gene; clone; bioinformatics analysis; tissue expression

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