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

Abstract

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

CLC Number:

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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Cloning and Analysis of FTO Gene of Nubian Goat and Construction of Eukaryotic Expression Vector

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