中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (2): 440-450.doi: 10.16431/j.cnki.1671-7236.2023.02.002

• 生物技术 • 上一篇    下一篇

努比亚山羊UCP1基因克隆、生物信息学分析及组织表达研究

韦义荣1, 郑自华2, 张叁保1, 宋颖1, 蒋海玉1, 孙雯玥1, 程鹏飞1, 刘雨帆1, 邹剑伟1, 黄艳娜1, 潘艳3, 蒋钦杨1   

  1. 1. 广西大学动物科学技术学院, 南宁 530004;
    2. 广西大学农牧产业发展研究院, 南宁 530004;
    3. 广西农业职业技术大学, 南宁 530009
  • 收稿日期:2022-07-05 出版日期:2023-02-05 发布日期:2023-02-06
  • 通讯作者: 潘艳, 蒋钦杨 E-mail:panyan413@163.com;jiangqinyang@126.com
  • 作者简介:韦义荣,E-mail:2899358074@qq.com;郑自华,E-mail:403796596@qq.com
  • 基金资助:
    国家重点研发计划项目(2021YFD1100100);国家现代农业产业体系广西牛羊产业创新团队建设项目(nycytxgxcxtd-2021-09);广西重点研发计划项目(桂科AB20297012)

Cloning,Bioinformatics Analysis and Tissue Expression of UCP1 Gene in Nubian Goats

WEI Yirong1, ZHENG Zihua2, ZHANG Sanbao1, SONG Ying1, JIANG Haiyu1, SUN Wenyue1, CHENG Pengfei1, LIU Yufan1, ZOU Jianwei1, HUANG Yanna1, PAN Yan3, JIANG Qinyang1   

  1. 1. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
    2. Agricultural and Animal Husbandry Industry Development Research Institute, Guangxi University, Nanning 530004, China;
    3. Guangxi Vocational University of Agriculture, Nanning 530009, China
  • Received:2022-07-05 Online:2023-02-05 Published:2023-02-06

摘要: 【目的】试验旨在克隆努比亚山羊解偶联蛋白-1(uncoupling protein-1,UCP1)基因并进行生物信息学分析,检测其在努比亚山羊不同组织中的表达差异,为研究努比亚山羊UCP1基因功能及进一步解析其在脂肪代谢中的调节作用提供数据。【方法】以努比亚山羊皮下脂肪组织cDNA为模板,采用PCR扩增并克隆UCP1基因CDS区序列后,与其他物种进行相似性比对及系统进化树构建,并对UCP1蛋白进行生物信息学分析;利用实时荧光定量PCR方法检测UCP1基因在努比亚山羊心脏、肝脏、脾脏、肾脏、背最长肌、皮下脂肪、腹脂中的相对表达量。【结果】努比亚山羊UCP1基因CDS区全长918 bp,编码305个氨基酸。相似性比对发现,努比亚山羊UCP1基因氨基酸序列与绵羊、瘤牛×普通牛、水牛、羚羊、马鹿、双峰驼、驴、大熊猫、人的相似性分别为98.1%、97.0%、96.5%、96.1%、95.8%、91.0%、87.0%、86.5%和83.8%。系统进化树表明,努比亚山羊与绵羊亲缘关系最近,与人的亲缘关系最远。生物信息学分析表明,努比亚山羊UCP1蛋白的分子质量为32.97 ku,等电点为9.29,属于碱性蛋白;UCP1蛋白缺乏稳定性,总平均亲水性为0.20,具备一定亲水性;脂溶性系数为91.70,跨膜螺旋氨基酸残基数量的预测值为15.94,前60个氨基酸的跨膜螺旋数预测值为8.76,位于膜细胞质侧的总概率为29.03%,属于非分泌蛋白;UCP1蛋白二级结构由α-螺旋(48.85%)、无规则卷曲(27.54%)、延伸链(16.39%)、β-转角(7.21%)构成;UCP1蛋白含有28个磷酸化位点、5个O-糖基化位点及2个N-糖基化潜在位点。实时荧光定量PCR结果显示,UCP1基因在努比亚山羊皮下脂肪中表达量最高,显著高于其他组织(P<0.05);在背最长肌中表达量最低。【结论】试验成功扩增UCP1基因CDS区序列,UCP1是一个缺乏稳定性、亲水的碱性蛋白;UCP1基因在努比亚山羊各组织中均有表达,且主要在皮下脂肪及脾脏中表达。结果为后续开展UCP1基因在脂肪产热供能中的机制研究提供了理论依据。

关键词: 努比亚山羊; UCP1基因; 克隆; 生物信息学分析; 组织表达

Abstract: 【Objective】 This study was aimed to clone uncoupling protein-1 (UCP1) gene in Nubian goats and carried out bioinformatics analysis,and detect its expression difference of UCP1 gene in different tissues of Nubian goats,in order to provide data for studying its regulatory function in fat metabolism and analyzing the function of UCP1 gene in Nubian goats.【Method】 Using the cDNA from the subcutaneous fat tissue in Nubian goats as a template,the CDS region of UCP1 gene in Nubian goats was amplified by PCR and cloned,sequence similarity alignment and phylogentic tree contruction were carried out with other species,and the bioinformatics analysis of UCP1 protein was carried out.The expression of UCP1 gene in heart,liver,spleen,kidney,longissimus dorsi muscle,subcutaneous fat and abdominal fat of Nubian goats were detected by Real-time quantitative PCR.【Result】 The CDS sequence of UCP1 gene in Nubian goats was 918 bp and encoded 305 amino acids.The similarity of amino acid sequence of UCP1 gene between Nubian goats and Ovis aries, Bos indicus× Bos taurus, Bubalus bubalis, Oryx dammah, Cervus elaphus, Canelus ferus, Equus asinus, Aliuropoda melanoleuca and Homo sapiens were 98.1%,97.0%,96.5%,96.1%,95.8%,91.0%,87.0%,86.5% and 83.8%,respectively.The phylogenetic tree indicated that the closest was to Ovis aries and the furthest was to Homo sapiens.The results of bioinformatics analysis showed that the molecular mass of UCP1 protein in Nubian goats was 32.97 ku,with an isoelectric point of 9.29,which was a basic proteins.UCP1 protein lacked stability,an overall average hydrophilicity was 0.20,which possessed certain hydrophilicity.The lipid solubility coefficient was 91.70,the predicted value of the number of amino acid residues in transmembrane helices was 15.94,the predicted value of the number of transmembrane helices in the first 60 amino acids of the protein was 8.76,the overall probability of being located on the cytoplasmic side of the membrane was 29.03%,and it belonged to nonsecretory proteins.The secondary structure was composed of alpha helix (48.85%),random coil (27.54%),extended chain (16.39%),and beta turn (7.21%).UCP1 had a total of 28 phosphate sites,5 potential O-glycosylation sites and 2 N-glycosylation potential sites.Real-time quantitative PCR results showed that the expression of UCP1 gene in subcutaneous fat of Nubian goats was relatively high,which was significantly higher than other tissues (P<0.05),while the expression of longissimus dorsi muscle in Nubian goats was the lowest.【Conclusion】The CDS sequence of UCP1 gene was successfully amplified,UCP1 was a basic protein lacking stability and hydrophilicity. UCP1 gene was widely expressed in various tissues of Nubian goats, which was mainly expressed in subcutaneous fat and spleen.The results provided a theoretical basis for subsequent research on the mechanism of UCP1 gene in the energy supply of fat thermogenesis.

Key words: Nubian goats; UCP1 gene; cloning; bioinformatics analysis; tissue expression

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