努比亚山羊脂肪和肥胖相关蛋白基因克隆分析及真核表达载体构建

doi:10.16431/j.cnki.1671-7236.2021.07.008

摘要/Abstract

摘要： 研究旨在对努比亚山羊脂肪和肥胖相关蛋白（fat mass and obesityassociated protein，FTO）基因进行克隆和分析，并构建其真核表达载体。取努比亚山羊背最长肌组织作为试验材料，采用RT-PCR扩增出FTO基因的编码区，测序鉴定后对得到的序列用相应的分析软件进行生物信息学分析，然后将FTO基因片段与pMD19-T载体连接后转化大肠杆菌DH5α感受态细胞，构建pMD19-T-FTO载体，测序正确的重组质粒双酶切后，连接pEGFP-N1载体构建pEGFP-N1-FTO真核表达载体，然后转染3T3-L1细胞，培养48 h后，在荧光显微镜下观察细胞表达荧光的情况。结果表明，试验成功克隆了努比亚山羊FTO基因的编码区，序列长度为1 518 bp，编码505个氨基酸，分子质量为57 142.24 u。努比亚山羊FTO基因编码区序列与NCBI上公布的山羊、牛、绵羊、猪、原鸡、小鼠的相似性分别为98.7%、96.4%、98.3%、87.2%、64.3%、81.7%，该基因系统进化树分析显示，努比亚山羊与山羊遗传距离最近，与原鸡遗传距离最远，在不同物种中具有高度保守性。努比亚山羊FTO蛋白二级和三级结构以α-螺旋和无规则卷曲为主。构建的真核表达载体pEGFP-N1-FTO转染3T3-L1细胞48 h后，在显微镜下观察到绿色荧光的表达，说明FTO基因真核表达载体构建成功。本试验构建努比亚山羊FTO基因真核表达载体，其在3T3-L1细胞中成功表达，为以后研究FTO基因与山羊脂肪代谢的相关性奠定基础。

关键词: 努比亚山羊; FTO基因; 克隆; 重组质粒; 真核表达载体

Abstract: This study was to clone and analyze the fat mass and obesityassociated protein (FTO) gene of Nubian goat,and construct its eukaryotic expression vector.The tissue of longissimus dorsi muscle of Nubian goat was taken as the experimental material,and the coding region of FTO gene was amplified by RT-PCR.After sequencing and identification,the cloned sequence was analyzed by bioinformatics with the corresponding analysis software,and then the FTO gene fragment was linked with pMD19-T vector and transformed into Escherichia coli DH5α competent cells.The pMD19-T-FTO vector was constructed,and the recombinant plasmid with correct sequencing was digested with double enzymes.The pEGFP-N1 vector was connected to construct the pEGFP-N1-FTO eukaryotic expression vector.The pEGFP-N1-FTO eukaryotic expression vector was then transfected into 3T3-L1 cells and cultured for 48 h.The results showed that the encoding region of Nubian goat FTO gene was successfully cloned.The sequence length was 1 518 bp,encoding 505 amino acids,and the molecular weight was 57 142.24 u.The homology of FTO gene of Nubian goat was 98.7%,96.4%,98.3%,87.2%,64.3% and 81.7% with that of Capra,Bos Taurus,Ovis aries,Sus scrofa,Gallus gallus and Mus musculus published on NCBI,respectively.The phylogenetic tree analysis of this gene showed that Nubian goat had the closest genetic distance to Capra and the furthest genetic distance to Gallus gallus.It was highly conserved in different species.The secondary and tertiary structures of Nubian goat FTO protein were mainly alpha helix and random coil.The constructed eukaryotic expression vector pEGFP-N1-FTO was transfected into 3T3-L1 cells 48 h later,and the expression of green fluorescence was observed under the microscope,indicating that the eukaryotic expression vector of FTO gene was successfully constructed.In this study,eukaryotic expression vector of Nubian goat FTO gene was constructed and successfully expressed in 3T3-L1 cells,which laid a foundation for future research on the correlation between FTO gene and goat fat metabolism.

Key words: Nubian goat; FTO gene; cloning; recombinant plasmid; eukaryotic expression vector

中图分类号:

邹菊红, 申玉建, 高小童, 黄艳娜, 蒋钦杨. 努比亚山羊脂肪和肥胖相关蛋白基因克隆分析及真核表达载体构建[J]. 中国畜牧兽医, 2021, 48(7): 2342-2348.

ZOU Juhong, SHEN Yujian, GAO Xiaotong, HUANG Yanna, JIANG Qinyang. Cloning and Analysis of FTO Gene of Nubian Goat and Construction of Eukaryotic Expression Vector[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2342-2348.

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