金黄地鼠Kcnq1基因的分子克隆与鉴定以及在多种器官组织中的表达差异分析  

Molecular cloning and identification of Kcnq1 gene andits expression differences in a variety of organs in golden hamsters

作　　者：孔德龙[1] 潘晓燕[1,2] 秦莹[1] 殷玉鹏[3] 安培培[1] 陈玥[1] 唐博[1] 李子义[1] 

机构地区：[1]吉林大学畜牧兽医学院动物胚胎工程吉林省重点实验室,吉林长春130062 [2]吉林医药学院,吉林吉林市132013 [3]西北农林科技大学,陕西杨凌712100

出　　处：《中国兽医学报》2012年第10期1521-1525,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金青年基金资助项目(30901056);博士点新教师基金资助项目(20090061120010)

摘　　要：对金黄地鼠Kcnq1基因进行分子克隆与鉴定以及在多种器官组织的表达差异进行分析,旨在研究Kcnq1基因在地鼠各组织器官中的功能。提取金黄地鼠心脏组织总RNA,根据大鼠Kcnq1的保守序列区域设计引物TK1,用RT-PCR的方法从金黄地鼠心脏组织中扩增出Kcnq1cDNA片段,并且以心脏cDNA片段为模板,将纯化后的cDNA在T4连接酶的作用下与pMD18-T载体特异性连接,转化感受态大肠杆菌DH5α中,筛选重组子并酶切鉴定,将鉴定后的重组子进行DNA测序。提取心、肝、脾、肺、肾各组织总RNA,并反转录,将各组织cDNA做荧光定量检测,检测各组织表达量的差异。结果显示克隆出金黄地鼠Kcnq1基因477bp的部分片段长度,推测出编码的159个氨基酸。与大鼠等物种Kcnq1基因比对,核苷酸和氨基酸序列均具有较高的同源性。荧光定量结果显示,Kcnq1在金黄地鼠心脏中表达量最高,在肺脏和肾脏中均有较高表达,在脾脏中低度表达,在肾脏中基本不表达。该研究结果为深入研究金黄地鼠Kcnq1基因功能奠定了基础。In order to investigate Kcnq1 gene function in golden hamsters,Kcnq1 gene was cloned and identified,and its expression differences was analysed in this study.Total RNA was extracted from golden hamster heart and primer TK1 was designed according to the rats Kcnq1 conservative sequence.Kcnq1 cDNA fragment was amplified from the heart using RT-PCR method.The purified cDNA was specifically linked with pMD18-T vector using the heart cDNA fragment as a template,which was then transformed into competent E.coli DH5α.After that,the recombinant was screened and identified and DNA sequencing.Total RNA was extracted from golden hamster heart,liver,spleen,lung and kidney,and Kcnq1 cDNA fragment was amplified using RT-PCR,expression differences in a variety of organs were detected by Real time PCR.The golden hamster Kcnq1 gene was cloned and total length was 477 bp,which indicated that it encoded 159 amino acids.The nucleotide and amino acid sequences had high homology when compared with rat etc..Real time PCR results showed that Kcnq1 gene was highest highly expressed in the heart,lung and kidney,less expressed in the spleen,almost no expressed in the liver.The expression in the heart was the highest.This study laid the foundation for further investigation for Kcnq1 gene function in golden hamsters.

关 键 词：金黄地鼠 KCNQ1 分子克隆 表达差异 

分 类 号：S852.23[农业科学—基础兽医学]