摘要
本研究对金黄地鼠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DH5a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,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,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 Kcnql gene function in golden hamsters, Kcnql 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 Kcnql conservative sequence. Kcnql 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 DH5a. 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 Kcnql cDNA fragment was amplified using RT-PCR expression differences in a variety of organs were detected by real time PCR. The golden hamster Kcnql 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 Kcnql 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 Kcnql gene function in golden hamsters.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第5期680-683,共4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)
关键词
金黄地鼠
KCNQ1
分子克隆
表达差异
golden hamster
Kcnql
molecular clone, expression differences
