Fat-1基因载体构建及其在家鸡中的体外表达

doi:10.16431/j.cnki.1671-7236.2016.04.010

›› 2016, Vol. 43 ›› Issue (4): 921-927.doi: 10.16431/j.cnki.1671-7236.2016.04.010

Fat-1基因载体构建及其在家鸡中的体外表达

王颖¹, 顾真真¹, 郭若婷¹, 欧科鹏¹, 刘乙¹, 刘小军^1,2

1. 石河子大学动物科技学院, 石河子 832003;
2. 河南农业大学牧医工程学院, 郑州 450002

修回日期:2015-11-13 出版日期:2016-04-20 发布日期:2016-04-27
通讯作者: 刘小军 E-mail:xjliu2008@hotmail.com
作者简介:王颖(1989-),女,新疆昌吉人,硕士生,研究方向:动物遗传育种与繁殖,E-mail:wy331362758@126.com
基金资助:
石河子大学校级育种专项(gxjs2012-yz06)

Construction of Fat-1 Gene Vector and its Expression in vitro of Chicken

WANG Ying¹, GU Zhen-zhen¹, GUO Ruo-ting¹, OU Ke-peng¹, LIU Yi¹, LIU Xiao-jun^1,2

1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;
2. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China

Revised:2015-11-13 Online:2016-04-20 Published:2016-04-27

摘要/Abstract

摘要： 试验旨在构建一种适合于家禽体外表达的ω-3脂肪酸脱氢酶Fat-1基因的真核生物表达载体,为后续转基因鸡的生产奠定基础。根据家鸡密码子使用频率,对秀丽隐杆线虫Fat-1基因进行优化改造;将改造后的Fat-1基因(cFat-1)连接至pLL3.7表达载体,构建重组质粒pLL3.7-cFat-1;经PCR、酶切、序列分析等手段对重组质粒进行鉴定;用胰酶消化法培养鸡成纤维细胞,利用TurboFect^TM转染试剂将pLL3.7-cFat-1转染体外培养的成纤维细胞;通过RT-PCR检测和绿色荧光观察分析cFat-1基因在细胞中的表达。结果表明cFat-1基因在鸡成纤维细胞中高效转录并表达,成功构建了可在家禽体外表达cFat-1基因的特异性表达载体。本研究首次获得可在家禽体外表达cFat-1基因的转基因细胞系,证实了经基因改造后cFat-1基因可在家禽体外的高效转录和表达,为后续制备富含ω-3脂肪酸的转基因鸡奠定基础。

关键词: ω-3脂肪酸脱氢酶; 载体构建; 成纤维细胞; 基因表达; 转基因

Abstract: The aim of the experiment was to construct an eukaryotic expression vector which could effectively express ω-3 fatty acid desaturase Fat-1 gene in cells of chicken and laid the foundations for production of transgenic chicken in the future.Based on the codon usage frequency of poultry, nucleotide sequence of Fat-1 gene of Caenorhabditis elegans was optimized (designed as cFat-1), synthesized and connected to pLL3.7 vector.The recombinant plasmid was confirmed by PCR, enzyme digestion and sequence analysis.The chicken embryo fibroblast cells were isolated using standard pancreatic enzyme digestion method and then transfected with pLL3.7-cFat-1 vector by TurboFect^TM transfection reagents in vitro.The gene expression was verified by RT-PCR analysis and green fluorescent detection.The results showed that cFat-1 gene could transcript and express efficiently in chicken embryo fibroblast cells indicating that the Fat-1 gene vector was constructed successfully.It was the first time that transgenic chicken fibroblast cell line which expressed cFat-1 gene was established.This study laid the foundation for the generation of transgenic chicken that were rich in ω-3 fatty acid.

Key words: ω-3 fatty acid desaturase; vector construction; fibroblasts; gene expression; transgenosis

中图分类号:

Q786

王颖, 顾真真, 郭若婷, 欧科鹏, 刘乙, 刘小军. Fat-1基因载体构建及其在家鸡中的体外表达[J]. , 2016, 43(4): 921-927.

WANG Ying, GU Zhen-zhen, GUO Ruo-ting, OU Ke-peng, LIU Yi, LIU Xiao-jun. Construction of Fat-1 Gene Vector and its Expression in vitro of Chicken[J]. , 2016, 43(4): 921-927.

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Fat-1基因载体构建及其在家鸡中的体外表达

Construction of Fat-1 Gene Vector and its Expression in vitro of Chicken

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