【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...【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。展开更多
As one member of the Ras super family, Rheb is an upstream regulator of mTOR signaling pathway, which regulates the process of cell-growth, proliferation and differentiation. In order to study the relationship between...As one member of the Ras super family, Rheb is an upstream regulator of mTOR signaling pathway, which regulates the process of cell-growth, proliferation and differentiation. In order to study the relationship between Rheb and mTOR in Inner Mongolian Cashmere goat （Capra hircus） cells, Ras homolog enriched in brain （Rheb） gene eDNA was amplified by RT-PCR. It is 555 bp in length and includes the complete ORF encoding 184 amino acids （GenBank accession no. HM569224）. The full eDNA nucleotide sequence has a 99% identity with that of sheep, 98% with cattle and 93% with human while their amino acids sequence shares identity with 98, 97 and 97% of them, correspondingly. The bioinformatics analysis showed that Rheb has a Ras family domain, two casein kinase II phosphorylation sites, two ATP/GTP-binding sites motifA （P-loop）, a prenyl group binding site （CAAX box）. Tissue-specific expression analysis performed by semi- quantitative RT-PCR. The Rheb gene was expressed in all the tested tissues and the highest level ofmRNA accumulation was detected in brain, suggesting that Rheb played an important role in goat cells.展开更多
文摘【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。
基金supported by grant from the National Natural Science Foundation of China (30860191)the Major Projects for New Varieties of Genetically Modified Organisms, China (2008ZX08008-002)the Training Fund for the Basic Sciences of China(J0730648)
文摘As one member of the Ras super family, Rheb is an upstream regulator of mTOR signaling pathway, which regulates the process of cell-growth, proliferation and differentiation. In order to study the relationship between Rheb and mTOR in Inner Mongolian Cashmere goat （Capra hircus） cells, Ras homolog enriched in brain （Rheb） gene eDNA was amplified by RT-PCR. It is 555 bp in length and includes the complete ORF encoding 184 amino acids （GenBank accession no. HM569224）. The full eDNA nucleotide sequence has a 99% identity with that of sheep, 98% with cattle and 93% with human while their amino acids sequence shares identity with 98, 97 and 97% of them, correspondingly. The bioinformatics analysis showed that Rheb has a Ras family domain, two casein kinase II phosphorylation sites, two ATP/GTP-binding sites motifA （P-loop）, a prenyl group binding site （CAAX box）. Tissue-specific expression analysis performed by semi- quantitative RT-PCR. The Rheb gene was expressed in all the tested tissues and the highest level ofmRNA accumulation was detected in brain, suggesting that Rheb played an important role in goat cells.
