未知人Na(+)-K(+)-Exchanging ATPase α1亚基基因第一内含子的获得及鉴定

Acquirement and Identification of a Novel Intron 1 of Human Na(+) K(+) Exchanging ATPase α1 Subunit Gene

目的研究人Ｎａ（＋）－Ｋ（＋）－ＥｘｃｈａｎｇｉｎｇＡＴＰａｓｅα１亚单位基因胞外区约８０～１３０位氨基酸编码序列的未知基因组结构。方法采用聚合酶链反应（ＰＣＲ）方法对人基因组ＤＮＡ及ｃＤＮＡ文库进行扩增，限制性酶切分析扩增产物，并进行荧光测序，对测序结果进行同源性分析及剪接位点的搜索并对得到的核苷酸序列进行分析。结果人基因组ＤＮＡ和ｃＤＮＡ经扩增后分别得到８３３和１９５ｂｐ两种不同大小的片段Ｆｇ，Ｆｃ。分析序列发现Ｆｇ与Ｆｃ相比在１３８～７７５ｂｐ处含有一６３８ｂｐ的插入片段，此片段与ＧｅｎＢａｎｋ中的任何已知序列均无明显同源性。结论本研究发现了一段全新未知的人Ｎａ（＋）－Ｋ（＋）－ＥｘｃｈａｎｇｉｎｇＡＴＰａｓｅα１亚单位基因的内含子全序列，并得到了第一外显子同第二外显子之间的相对位置和序列，其在Ｇｅｎ－Ｂａｎｋ的基因检索号为Ｌ７６９３８。序列分析表明该内含子可能具有潜在的调控基因表达的功能，并含有一个可能的编码序列。

Objective The unknown part genome structure of the α1 subunit of human Na(+) K(+) Exchanging ATPase gene which covers the 80￣130 aa in its extracellular domain was studied. Methods By means of PCR, the targeted sequences was amplified by using two different templates: human Genome DNA and the muscle cDNA library; the PCR products were analyzed by restriction endonuclease digestion, then cloned into plasmid vector to perform the chemiluminescence Sequencing. The homologous search, splicing site detection and the analysis of the sequence were applied. Results Two different lengths of products, 833 bp(Fg) and 195 bp(Fc) were acquired after PCR amplification with the human genome DNA and muscle cDNA library respectively. There was an insertion of a 638 bp fragment in Fg at its 138￣775 bp. And there was not obviously known sequence homologous to the insertion sequence. Conclusions A novel sequence containing the complete human Na(+) K(+) Exchanging ATPase α1 subunit gene intron 1 was found, the exact splicing donor and receptor site within it were identified. The Accession No of this sequence is L76938. Analysis of this sequence showed that the intron 1 may function in the regulation of the gene expression and also had a potential coding sequence.