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

摘要/Abstract

摘要： 【目的】研究旨在对努比亚山羊大脑富集Ras同源物(Ras homolog enriched in brain,Rheb)基因进行克隆和分析,并构建其真核表达载体,为进一步揭示Rheb对努比亚山羊骨骼肌调控的分子机理奠定基础。【方法】试验采用RT-PCR方法从努比亚山羊背最长肌组织中扩增Rheb基因,经琼脂糖凝胶电泳及测序验证正确后进行生物信息学分析,同时构建该基因真核表达载体,转染细胞后进行实时荧光定量PCR检测来验证所构建载体的正确性。【结果】努比亚山羊Rheb基因编码区序列长度为555 bp,编码 184个氨基酸,Rheb蛋白分子式为C₉₁₀H₁₄₅₆N₂₃₆O₂₇₉S₆,分子质量为20 359.34 u,原子总数为2 887,理论等电点为5.93。Rheb蛋白属于稳定的亲水性蛋白,不包含跨膜结构域。蛋白二级结构预测结果显示,努比亚山羊Rheb蛋白中α-螺旋、β-转角、延伸链和无规则卷曲分别占40.76%、7.07%、22.83%和29.35%。构建的真核表达载体pcDNA3.1-Rheb转染山羊骨骼肌细胞后,与空载体组相比,Rheb基因表达量极显著升高(P＜0.01)。【结论】本试验成功克隆努比亚山羊Rheb基因编码区序列,并构建pcDNA3.1-Rheb真核表达载体,这为深入理解Rheb基因在努比亚山羊肌肉中的作用提供了理论支持。

关键词: 努比亚山羊; Rheb基因; 生物信息学; 骨骼肌细胞; 真核表达载体

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

中图分类号:

S813

邹菊红, 莫智化, 刘雨帆, 邹剑伟, 卢俊, 何海恩, 王璠, 黄艳娜, 蒋钦杨. 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[J]. 中国畜牧兽医, 2024, 51(5): 1819-1826.

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.

参考文献

[1] TAMAI T,YAMAGUCHI О,HIKOSO S,et al.Rheb (Ras homologue enriched in brain)-dependent mammalian target of rapamycin complex 1(mTORCl)activation becomes indispensable for cardiac hypertrophic growth after earlypostnatal period[J]. Journal of Biological Chemistry,2013,288(14):10176-10187.
[2] GOODMAN C A,MIU M H,FREYJ W,et al.A phosphatidylinositol 3-kinase/proteinkinase B-independent activation of mammalian target of rapamycin signaling issufficient to induce skeletal muscle hypertrophy[J]. Molecular Biology of the Cell,2010,21(18):3258-3268.
[3] GE Y,CHEN J.Mammalian target of rapamycin (mTOR) signaling network inskeletal myogenesis[J].Journal of Biological Chemistry,2012,287(52):43928-43935.
[4] ASHRAF S,HAN I B,PARK H,et al.Role of Rheb in regulating differentiation fate of mesenchymal stem cells for cartilage and bone regeneration[J].International Journal of Molecular Sciences, 2017,18(4):880-893.
[5] YANG W,PANG D,CHEN M,et al.Rheb mediates neuronal-activity-induced mitochondrial energetics through mTORC1-independent PDH activation[J].Developmental Cell, 2021,56(6):811-825.
[6] BLACKWOOD E A,HOFMANN C,SANTO DOMINGO M,et al.ATF6 regulates cardiac hypertrophy by transcriptional induction of the mTORC1 activator,Rheb[J].Circulation Research,2019,124(1):79-93.
[7] DENG L,CHEN L,ZHAO L,et al.Ubiquitination of Rheb governs growth factor-induced mTORC1 activation[J].Cell Research,2019,29(2):136-150.
[8] 高娟,杨爽,王玉霞,等.Rheb1在小鼠巨核-红系多能性干细胞发育中的作用[J].中国实验血液学杂志,2022,30(1):298-304. GAO J,YANG S,WANG Y X,et al.Effect of Rheb1 in the development of mouse megakaryocyte-erythroid progenitor cells[J].Journal of Experimental Hematology, 2022,30(1):298-304.(in Chinese)
[9] 窦露,刘畅,杨致昊,等.动物肌肉组织蛋白质代谢调控的研究进展[J].动物营养学报,2022,34(1):39-50. DOU L,LIU C,YANG Z H,et al.Research progress on regulation of protein metabolism in animal muscle tissue[J].Chinese Journal of Animal Nutrition,2022,34(1):39-50.(in Chinese)
[10] BODINE S C,STITT T N,GONZALEZ M,et al.Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo[J].Nature Cell Biology,2001,3(11):1014-1019.
[11] ZANCHI N E,L ANCHA A H J R.Mechanical stimuli of skeletal muscle:Implications on mTOR/p70 s6k and protein synthesis[J]. European Journal of Applied Physiology,2008,102(3):253-263.
[12] LIU Y,ZHONG W,ZHANG J,et al.Tetrandrine modulates Rheb-mTOR signaling-mediated selective autophagy and protects pulmonary fibrosis[J].Frontiers in Pharmacology,2021,12:739220.
[13] JIA L,LIAO M,MOU A,et al.Rheb-regulated mitochondrial pyruvate metabolism of Schwann cells linked to axon stability[J].Developmental Cell,2021,56(21):2980-2994.
[14] ASPURIA P J,TAMANOI F.The Rheb family of GTP-binding proteins[J].Cellular Signalling,2004,16(10):1105-1112.
[15] GROMOV P S,MADSEN P,TOMERUP N,et al.A novel approach for expression cloning of small GTPases:Identification,tissue distribution and chromosome mapping of the human homolog of Rheb[J].FEBS Letters,1995,377(2):221-226.
[16] MIZUKI N,KIMURA M,OHNO S,et al.Isolation of cDNA and genomic clones of a human ras-related GTP-binding protein gene and its chromosomal localization to the long arm of chromosome 7,7q36[J].Genomics,1996,34(1):114-118
[17] YANG W,PANG D,CHEN M,et al.Rheb mediates neuronal-activity-induced mitochondrial energetics through mTORC1-independent PDH activationr[J].Developmental Cell,2021,56(6):811-825.
[18] LAPLANTE M,SABATINI D M.mTOR signaling at a glancer[J].Journal of Cell Science,2009,122(Pt 20):3589-3594.
[19] RICHTER J D,SONENBERG N.Regulation of cap-dependent translation by eIF4E inhibitory proteinsr[J].Nature,2005,433(7025):477-480.
[20] GOODMAN C A,MIU M H,FREY J W,et al.A phosphatidylinositol 3-kinase/protein kinase B-independent activation of mammalian target of rapamycin signaling is sufficient to induce skeletal muscle hypertrophyr[J].Molecular Biology of the Cell,2010,21(18):3258-3268.
[21] LEE R C,WANG Z,HEO M,et al.Total-body skeletal muscle mass:development and cross-validation of anthropometric prediction models[J].The American Journal of Clinical Nutrition,2000,72(3):796-803.
[22] IZUMIYA Y,HOPKINS T,MORRIS C,et al.Fast/glycolytic muscle fiber growth reduces fat mass and improves metabolic parameters in obese micer[J]. Cell Metabolism,2008,7(2):159-172.
[23] 刘正平.养殖努比亚山羊发大财[J].湖南农业,2012,11:21. LIU Z P.Breeding Nubian goats to make a fortune[J].Hunan Agricuture,2012,11:21.(in Chinese)
[24] 王景圆.Rheb基因对绒山羊骨骼肌卫星细胞的影响[D].呼和浩特:内蒙古大学,2014. WANG J Y.The role of Rheb gene in cashmere goat satellite muscle cells[D].Hohhot:Inner Mongolia University,2014.(in Chinese)
[25] WILKINSON G W,AKRIGG A.Constitutive and enhanced expression from the CMV major IE promoter in a defective Adenovirus vector[J]. Nucleic Acids Research,1992,20(9):2233-2239.