藏羚羊ET-1基因编码区的克隆与分析  

MOLEULAR CLONING AND SEQUENCE ANALYZING OF ET-1 CODING cDNA ON TIBETAN ANTELOPE

作　　者：冯志超[1] 马兰[1] 白振忠[1] 格日力[1] 

机构地区：[1]青海大学高原医学研究中心

出　　处：《青海医学院学报》2012年第4期251-256,共6页Journal of Qinghai Medical College

基　　金：973计划项目(NO:2012CB518200)

摘　　要：目的克隆藏羚羊内皮素1(endothelin-1,ET-1)基因编码区,并行分析。方法从藏羚羊肺组织中提取总RNA,通过RT-PCR法获得藏羚羊ET-1cDNA,将其与pGEM-T Easy载体连接构建重组质粒,经转化JM109菌扩增培养后鉴定阳性质粒并测序。将测序结果与NCBI数据库进行同源性比较。结果获得藏羚羊ET-1基因编码区序列,长度为609bp,编码202个氨基酸,将此序列提交GenBank数据库并取得注册号(JQ031153)。其核苷酸序列与绵羊、牛、猪、大鼠以及斑马鱼的同源性分别是:98%、95%、86%、81%、48%。推测出的氨基酸序列与绵羊、牛、猪、大鼠以及斑马鱼的同源性分别是:97%、94%、79%、76%、34%。与绵羊ET-1氨基酸比对发现,30位和172位的苏氨酸变为丙氨酸、40位和163位的缬氨酸变为丙氨酸、141位天冬氨酸变为天冬酰胺、181位的苯丙氨酸变为缬氨酸。结论成功克隆了藏羚羊肺组织中ET-1基因的编码区,分析发现藏羚羊ET-1基因对应的氨基酸发生了突变,这些为进一步揭示藏羚羊低氧适应分子机制打下基础。Objective To clone and analyze the encoding region of ET-1 gene from Tibetan antelope.Methods Total RNA was isolated from a Tibetan antelope lung tissue,and Tibetan antelope ET-1 gene was amplified by RT-PCR.The PCR product was cloned into pGEM-T vector and sequenced.Nucleotide sequences were compared with GenBank data by Blast method.Results The encoding region of ET-1 gene of Tibetan antelope,which is composed of 609 bp and codes 202 amino acids,was obtained and deposited in GenBank as accession number JQ031153.DNA sequencing proved that Tibetan Antelope ET-1 sequence was highly homologous with the following species such as sheep（96%）,cattle（96%）,pig（87%）,and rat（81%）.The deduced Amino acid sequence showed 97%,94%,79%,and 76% identity when compared with that of the sheep,cattle,pig,and rat.When comparing the ET-1 Sequence of Tibetan Antelope logined in NCBI Databank with that of sheep,six mutations can be observed at the corresponding amino acid sequence of 30 and 172（Thr→Ala）,40 and 163（Val→Ala）,141（Asp→Asn）,181（Phe→Val）.Conclusion The encoding region of Tibetan antelope ET-1 gene is successfully cloned,which provides basic information for elucidating the possible molecular mechanism in high altitude adaptation of Tibetan antelope.

关 键 词：CDNA 克隆 ET-1 藏羚羊 低氧适应 

分 类 号：Q781[生物学—分子生物学]