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  

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

作　　者：邹菊红 莫智化 刘雨帆 邹剑伟 卢俊 何海恩 王璠 黄艳娜[1] 蒋钦杨[1] ZOU Juhong;MO Zhihua;LIU Yufan;ZOU Jianwei;LU Jun;HE Haien;WANG Fan;HUANG Yanna;JIANG Qinyang(College of Animal Science and Technology,Guangxi University,Nanning 530000,China)

机构地区：[1]广西大学动物科学技术学院,南宁530000

出　　处：《中国畜牧兽医》2024年第5期1819-1826,共8页China Animal Husbandry & Veterinary Medicine

基　　金：广西研究生教育创新计划项目(YCBZ2023016);国家现代农业产业体系广西牛羊产业创新团队建设项目(nycytxgxcxtd-2021-09)。

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C 910 H 1456 N 236 O 279 S 6,分子质量为20359.34 u,原子总数为2887,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。【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 C 910 H 1456 N 236 O 279 S 6,the molecular weight was 20359.34 u,the total number of atoms was 2887,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.

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 

分 类 号：S813[农业科学—畜牧学]