牦牛Endothelin-1基因CDS序列克隆及其真核表达质粒构建

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609bp的开放阅读框，编码202个氨基酸，该蛋白分子量为22.98 kDa，理论等电点（pI）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pEGFP-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。

Cloning of the CDS of yak Endothelin-1 gene and construction ofits eukaryotic expression plasmid

Endothelin-1 is a potent vasoconstricting factor that can induce structural and functional changes of the blood vessel by promoting vascular smooth muscle cell proliferation. This study was designed to study the molecular basis of high-altitude adaption in yak with a focus on the potential role of Endothelin-1. We cloned and analyzed the CDS of yak (Bos grunniens) Endothelin-1 and constructed a eukaryotic expression plasmid to provide a useful tool to study its fuction in the future. The results showed that the full-length of yak Endothelin-1 contains a complete ORF (609bp) encoding 202 amino acids. The putative molecular weight and theory isoelectric point of Endothelin-1 gene in yak are 22.98 kDa and 9.63. We also successfully constructed a eukaryotic expression vector pEGFP-C1-Endothelin-1 in which the CDS of the Endothelin-1 is driven by CMV promoter and the expression can be monitored by GFP. Amino acid sequences of yak Endothelin-1 are 100%、93%、81%、77%、70% identical to those in Bos Taurus、Ovis aries、Pantholops hodgsoni and Homo sapiens, respectively. In summary, findings from the study will pave the way to further explore the molecular mechanism of high-altitude adaption in yak and likely other animals.