努比亚山羊Rheb基因克隆、生物信息学分析及真核表达载体构建

doi:10.16431/j.cnki.1671-7236.2024.05.003

Abstract

Abstract: 【Objective】 The aim of this study was to clone and analyze Ras homolog enriched in brain (Rheb) gene in Nubian goats,and construct its eukaryotic expression vector,which laid a foundation for further revealing the molecular mechanism of Rheb regulation on the skeletal muscle of Nubian goats.【Method】 Rheb gene was amplified from the longissimus dorsi muscle tissue of Nubia goats by RT-PCR method.After being verified by agarose gel electrophoresis and sequencing,the bioinformatics analysis was carried out.At the same time,the eukaryotic expression vector of Rheb gene was constructed.After transfecting cells,Real-time quantitative PCR was performed to verify the correctness of the constructed vector.【Result】 The length of coding region of Rheb gene in Nubian goats was 555 bp,encoding 184 amino acids.The molecular formula of Rheb protein was C₉₁₀H₁₄₅₆N₂₃₆O₂₇₉S₆,the molecular weight was 20 359.34 u,the total number of atoms was 2 887,and the theoretical isoelectric point was 5.93.Rheb protein was found to be a stable hydrophilic protein,which did not contain transmembrane domains.The results of protein secondary structure prediction showed that alpha helix,beta turn,extended chain and random coil accounted for 40.76%,7.07%,22.83% and 29.35%,respectively,in Rheb protein of Nubian goats.After transfecting the constructed eukaryotic expression vector pcDNA3.1-Rheb into goat skeletal muscle cells,the expression of Rheb gene was extremely significantly increased compared with the empty vector group (P＜0.01).【Conclusion】 This experiment successfully cloned the sequence of the coding region of Rheb gene and constructed the eukaryotic expression vector pcDNA3.1-Rheb,which provided theoretical support for the in-depth understanding of the role of Rheb gene in the muscle of Nubian goats.

Key words: Nubian goats; Rheb gene; bioinformatics; skeletal muscle cells; eukaryotic expression vector

CLC Number:

S813

ZOU Juhong, MO Zhihua, LIU Yufan, ZOU Jianwei, LU Jun, HE Haien, WANG Fan, HUANG Yanna, JIANG Qinyang. Cloning,Bioinformatics Analysis and Eukaryotic Expression Vector Construction of Rheb Gene in Nubian Goats[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1819-1826.

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

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