努比亚山羊LMCD1基因生物信息学分析、真核表达载体的构建及表达

doi:10.16431/j.cnki.1671-7236.2022.06.003

Abstract

Abstract: 【Objective】 The purpose of this study was to amplify LIM domain gene 1 (LMCD1) in Nubian goats and analyze using bioinformatics, construct eukaryotic expression vector, and detect the expression of LMCD1 gene, so as to provide a reference for studying the function of LMCD1 gene in Nubian goats and exploring the role of LMCD1 gene in skeletal muscle development of goats.【Method】 Total RNA was extracted from the longissimus dorsi muscle in Nubian goats.The CDS region of LMCD1 gene in Nubian goats was amplified by RT-PCR and analyzed by bioinformatics, and LMCD1 gene was connected to pEGFP-N1 vector by homologous recombination.After enzyme digestion and sequencing identification, the positive plasmid was named pEGFP-N1-LMCD1 recombinant plasmid.pEGFP-N1-LMCD1 recombinant plasmid was transfected into skeletal muscle satellite cells in goats, the expression of LMCD1 gene in Nubian goats was detected by Real-time quantitative PCR.【Result】 The total length of LMCD1 gene CDS in Nubian goats was 1 092 bp, encoding 363 amino acids.The molecular formula of LMCD1 protein was C₁₇₇₅H₂₈₁₈N₅₀₈O₅₃₃S₂₉, and the molecular weight was 40.73 ku.The sequence of LMCD1 gene CDS in Nubian goats had the highest similarity with Capra hircus (99.8%) and the lowest similarity with Danio rerio (55.4%), and the similarity with other species was 87.0% to 98.8%.LMCD1 protein had no signal peptide and transmembrane domain, and was a hydrophilic protein.pEGFP-N1-LMCD1 eukaryotic expression vector in Nubian goats was constructed and transfected into skeletal muscle satellite cells.LMCD1 gene was overexpressed to produce green fluorescence signal.【Conclusion】 In this study, LMCD1 gene CDS in Nubian goats was amplified successfully, the pEGFP-N1-LMCD1 eukaryotic expression vector was constructed, and its biological function was analyzed, which provided a theoretical basis for the follow-up study of the mechanism of LMCD1 gene in skeletal muscle development of goats.

Key words: Nubian goats; LMCD1 gene; bioinformatics analysis; eukaryotic expression vector; expression

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ZOU Jianwei, ZOU Juhong, SHEN Yujian, WEI Yi, ZHANG Sanbao, LIAN Zitong, XU Jianjian, SONG Ying, HUANG Yanna, WEI Yingming, JIANG Qinyang, ZHENG Zihua. Bioinformatics Analysis, Construction of Eukaryotic Expression Vector and Expression of LMCD1 Gene in Nubian Goats[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2033-2042.

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Bioinformatics Analysis, Construction of Eukaryotic Expression Vector and Expression of LMCD1 Gene in Nubian Goats

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