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

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。

The aim of the experiment was to construct an eukaryotic expression vector which could effectively expressω-3fatty acid desaturase Fat-1gene 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-1gene of Caenorhabditis elegans was optimized（designed as cFat-1）,synthesized and connected to pLL3.7vector.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-1vector by TurboFect TMtransfection reagents in vitro.The gene expression was verified by RT-PCR analysis and green fluorescent detection.The results showed that cFat-1gene could transcript and express efficiently in chicken embryo fibroblast cells indicating that the Fat-1gene vector was constructed successfully.It was the first time that transgenic chicken fibroblast cell line which expressed cFat-1gene was established.This study laid the foundation for the generation of transgenic chicken that were rich inω-3fatty acid.