大菱鲆钙激活型钾离子通道基因TSKCa的克隆和序列分析

Cloning and Sequence Analysis of Calcium Activated Potassium Channel Gene From Turbot(Scophthalmus maximus L.)

钙激活型钾离子通道蛋白（KCa）是位于细胞膜表面调节细胞功能的一类膜蛋白。通过设计特异性引物，以我国海水养殖大菱鲆（Scophthal musmaximus L．）血液cDNA为模板，利用RT-PCR技术克隆获得了一条基因片段。生物信息学分析证实该片段来自钙激活型钾离子通道基因SKCa（小电导钙激活型钾通道）家族。设计巢式PCR引物，通过5′RACE和3′RACE分别克隆得到了该基因的5′端序列和3′端序列。使用生物学软件拼接3段序列得到了大菱鲆钙激活型钾离子通道TSKCa基因的全长序列。TSKCa基因cDNA全长为1698bp，其中开放阅读框长度为1632bp，编码的TSKCa蛋白有544个氨基酸。比对TSKCa蛋白与GenBank中已有的SKCa蛋白序列，证实其序列同源性很高。使用网络DAS和srsbin服务器对TSKCa蛋白进行跨膜结构预测和疏水性分析，并结合已有的研究结果推测TsKCa蛋白的结构由S1-S6六个跨膜结构域和一个孔道区（P区）组成。

The calcium-activated potassium channel （KCa） protein is a kind of membrane protein located on the cell membrane for regulating cell functions. A set of primers was designed and RT-PCR technique was used to obtain a gene fragment by taking the turbot blood cDNA as a template, and the gene fragment was indentified as a small conductance calcium activated potassium channel （SKCa） gene. The 5＇end sequence and 3＇end sequence of the gene fragment were obtained respectively by using 5＇RACE and 3＇RACE tech-niques, and the 3 gene sequences were assembled to obtain the full-length sequence of TSKCa gene of turbot calcium activated potassium channel gene. The full length of TSKCa gene is 1698 bp, the length of open reading frame is 1632 bp, and there are 544 amino acids in the coded TSKCa protein. It is shown from the comparison between turbot TSKCa protein and SKCa proteins of other species in GenBank that turbot TSKCa protein is highly homologous to SKCa proteins of other species. The trans-membrance structure prediction and hydrophobicity analysis of turbot TSKCa protein were made by using web DAS server and srsbin server, and it is inferred from the analysis results that TSKCa protein has an ion channel structure consisting of a pore region and 6 trans-membrane domains of S1 to S6.