鸡Dazl基因的克隆及其亚细胞定位研究  

Cloning and Subcellular Localization of Dazl Gene in Chicken

作　　者：郑蒙蒙[1] 李伟[1] 施青青[1] 王丹[1] 黄晓梅[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009

出　　处：《中国家禽》2013年第6期8-12,共5页China Poultry

基　　金：国家自然科学基金资助项目(30871791);江苏省研究生培养科研创新计划项目(2010-05);江苏省"六大人才高峰"项目;江苏省高校自然科学重大基础研究项目(08KJA230001)

摘　　要：为克隆鸡Daz(lDeleted in azoospermia-like,Dazl)基因CDS序列,通过构建eGFP标记的DAZL真核表达载体,实现该基因产物的亚细胞定位以探究其生物信息学功能。提取1日龄鸡睾丸总RNA,通过巢式PCR方法扩增出Dazl基因的CDS,构建真核表达载体pEGFP-C1-DAZL。采用LipofectaminTMLTX介导重组表达载体pEGFP-C1-DAZL转染CEF细胞,48h后于荧光倒置显微镜下观察其表达定位,同时利用RT-PCR和Western-blot进一步鉴定eGFP-Dazl和蛋白水平的表达情况。结果表明:克隆出Dazl基因的完整CDS,长度870bp,共编码289个氨基酸,与公布的鸡Dazl基因(GenBank登陆号:NM_204218)CDS区同源性达99%,编码氨基酸完全一致。酶切鉴定和序列分析均表明真核表达载体pEGFP-C1-DAZL构建成功。转染48h后,RT-PCR和Western-blot分别检测到949bp特异条带和59.7ku的融合蛋白。荧光显微镜观察显示,融合蛋白(eGFP-Dazl)主要分布于细胞核。The aim of the study was to clone the CDS of Dazl gene and to analyze its subcellular localization through eGFP fusion protein. CDS of Dazl gene was cloned by nested PCR from eDNA, and fusion expression vector named pEGFP-C1-DAZL containing enhanced green fluorescent protein （eGFP）was constructed to visualize the DAZL under flourescenee, pEGFP-C1-DAZL was transfected into CEF cells, using LipofeetaminTM LTX,48 h later, the Dazl-eGFP was observed under fluorescence inverted microscope. The mRNA and protein expression in vitro was detected using RT-PCR and Western-blot methods. The results showed that DAZL CDS was successfully cloned and the complete open reading frame size was 870 bp,and encode 289 amino acids. And the cloned CDS was 99% in homology with the published sequence in GenBank, while amino acids it encoded was exactly the same. The eukaryotic expression vectorpEGFP-C1-DAZL was constructed successfully, after 48 h transfected into CEF, the DAZL-eGFP fusion protein was observed localized at the nucleus significantly. The eukaryotic expression vector recombinant plasmid pEGFP-C1-DAZL was successfully constructed,with expression ability.

关 键 词：鸡 DAZL 真核表达载体 绿色荧光蛋白 亚细胞定位 

分 类 号：S852.659.5[农业科学—基础兽医学]