斑马鱼Gfi1.1基因的克隆及其体外转录  

Cloning of Zebrafish Gfi1.1 cDNA and Its in Vitro Transcription

作　　者：朱贤敏[1] 姜利军[1] 赵磊[1] 关军[1] 尹琎[1] 张义成[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院血液科,湖北武汉430030

出　　处：《现代生物医学进展》2014年第10期1818-1820,1812,共4页Progress in Modern Biomedicine

摘　　要：目的:克隆斑马鱼Gfi1.1基因的全长cDNA,运用T7 RNA聚合酶对含有Gfi1.1基因的ORF区进行体外转录,在体外合成5端带有帽子结构的Gfi1.1 mRNA分子,为后续研究斑马鱼Gfi1.1基因的功能打下基础。方法:应用RT-PCR从斑马鱼组织中扩增出Gfi1.1 cDNA片段,经回收纯化与pGM-T载体连接并转化感受态细菌DH-5α,通过蓝白筛选酶切鉴定阳性菌落,小量提取质粒,Nde I限制性内切酶线性化pGM-T-Gfi1.1质粒,运用T7 RNA聚合酶对Gfi1.1基因进行体外转录及加帽。经凝胶电泳对目的片段进行鉴定。结果:RT-PCR扩增获得约1.2 kb的DNA片段,DNA序列分析的结果与GenBank上的序列(NM_001020776)一致,酶切线性化及体外转录加帽pGM-T-Gfi1.1,凝胶电泳鉴定RNA分子大小与预期完全一致。结论:成功克隆斑马鱼Gfi1.1基因并体外转录及加帽pGM-T-Gfi1.1。Objective： To clone the full-length of zebrafish Gfil. 1 cDNA,transcript the Open Reading Frame region of the Gfil. 1 gene in vitro by T7 RNA Polymerase, synthesize a Gill.1 mRNA molecule capped at its 5 terminal and provide a basis to further study the biological functions of zebrafish Gill. 1. Methods： Total RNA was isolated from the tissues of zebrafish, and the full-length Gill. 1 cDNA was amplified by RT-PCR and then ligated to pGM-T vector after retrieve and purification. The product was transformed into competent cells DH-5ct. The positive recombinant clones were selected and identified by the complementation, restriction endonuclease digestion. After the plasmid pGM-T-Gfil.1 extraction and linearization by Nde I, gene gfil.1 was transcripted in vitro by T7 RNA Polymerase and capped with the capped analog. Then the product was identified. Results： A fragment of 1.2 kb was gained by RT-PCR and its sequence was identical to the sequence deposited in GenBank （NM_001020776）. After restriction endonuclease linearization and transcription in vitro, the gill. 1 RNA product proved to be consistent with the expected results by gel electrophoresis. Conclusion： The zebrafish Gene Gill. 1 is successfully cloned and transcripted in vitro.

关 键 词：Gfi1 1基因 克隆 体外转录 

分 类 号：Q78[生物学—分子生物学]