| 朱贤敏姜利军赵磊关军尹琎张义成△.斑马鱼Gfi1.1 基因的克隆及其体外转录[J].现代生物医学进展英文版,2014,14(10):1818-1820. |
| 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 |
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| DOI： |
| 中文关键词: Gfi1.1 基因 克隆 体外转录 |
| 英文关键词: Gfi1.1 gene Cloning Transcript in vitro |
| 基金项目: |
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| 中文摘要: |
| 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-5琢，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。 |
| 英文摘要: |
| ABSTRACT Objective：To clone the full-length of zebrafish Gfi1.1 cDNA,transcript the Open Reading Frame region of the Gfi1.1
gene in vitro by T7 RNA Polymerase, synthesize a Gfi1.1 mRNA molecule capped at its 5 terminal and provide a basis to further study
the biological functions of zebrafish Gfi1.1. Methods：Total RNA was isolated from the tissues of zebrafish, and the full-length Gfi1.1
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-5琢. The positive recombinant clones were selected and identified by the complementation, restriction endonuclease
digestion. After the plasmid pGM-T-Gfi1.1 extraction and linearization by Nde I, gene gfi1.1 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 gfi1.1 RNA product proved to be consistent with the expected results by gel electrophoresis.Conclusion： The
zebrafish Gene Gfi1.1 is successfully cloned and transcripted in vitro. |
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