牦牛Endothelin-1基因CDS序列克隆及其真核表达质粒构建

发布时间：2018-05-24 15:00

本文选题：牦牛 + 内皮素　；参考：《兽类学报》2017年03期

【摘要】：Endothelin-1主要通过促进血管平滑肌细胞增殖引起血管结构和功能的改变,从而刺激血管收缩,Endothelin-1在动物血管生成和低氧适应等关键发育和生理过程中发挥重要的作用。为了进一步探讨高原动物脑Endothelin-1对其极端低氧适应调控的分子机理,本研究对牦牛(Bos grunniens)脑Endothelin-1基因CDS全长序列进行了克隆及其真核表达载体的构建。结果表明,牦牛Endothelin-1基因的CDS全长序列含有一个609 bp的开放阅读框,编码202个氨基酸,该蛋白分子量为22.98 k Da,理论等电点(p I)为9.63;成功构建出带有CMV启动子,绿色荧光蛋白标记的牦牛Endothelin-1真核表达质粒p EGFP-C1-Endothelin-1;牦牛Endothelin-1基因CDS全长序列编码的氨基酸序列与黄牛(Bos taurus)、藏羚羊(Pantholop shodgsoni)、绵羊(Ovis aries)、野猪(Sus scrofa)、小鼠(Mus musculus)、人(Homo sapiens)的同源性分别为100%、95%、93%、81%、77%、70%;且该氨基酸序列的系统进化情况与其亲缘关系远近一致。因此,我们认为高原动物牦牛Endothelin-1的结构和功能在进化上均具有高度保守性,其在低氧适应中的作用可能是通过表达差异或转录后修饰实现的,且本研究构建的真核表达质粒为进一步探讨该基因在青藏高原动物脑对极端低氧生境适应中的生物学功能及其调控机理提供了支持。
[Abstract]:Endothelin-1 plays an important role in the development and physiological process of angiogenesis and hypoxic adaptation by promoting the proliferation of vascular smooth muscle cells and causing the changes of vascular structure and function, thus stimulating the vasoconstriction of Endothelin-1. In order to further investigate the molecular mechanism of brain Endothelin-1 regulating extremely hypoxic adaptation in plateau animals, the full-length CDS sequence of the brain Endothelin-1 gene of Bos grunniens was cloned and its eukaryotic expression vector was constructed. The results showed that the full-length CDS sequence of yak Endothelin-1 gene contained a 609bp open reading frame encoding 202 amino acids, the molecular weight of the protein was 22.98kDaand the theoretical isoelectric point (Ip) was 9.63. the promoter with CMV was successfully constructed. The eukaryotic expression plasmid pEGFP-C1-Endothelin-1of yak Endothelin-1 labeled with green fluorescent protein; the amino acid sequence encoded by the full-length CDS sequence of yak Endothelin-1 gene is homologous to Bos taurusus, Pantholop shodgsonike, Ovis ariesus, Sus scrofaa, Mus musculus, Homo sapiensus, respectively. The phylogeny of the amino acid sequence is consistent with its phylogenetic relationship. Therefore, we believe that the structure and function of Endothelin-1 in plateau animals are highly conserved in evolution, and its role in hypoxic adaptation may be achieved by expression difference or post-transcriptional modification. The eukaryotic expression plasmid was constructed in this study to further study the biological function and regulation mechanism of the gene in the animal brain of Qinghai-Xizang Plateau to adapt to extremely hypoxic habitat.
【作者单位】：兰州大学生命科学学院动物学与发育生物学研究所;西北师范大学生命科学学院;
【基金】：国家自然科学基金项目(31000190,051000006,31060141)
【分类号】：Q953

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