【作者】 武珅；

【导师】 王宗礼；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 n-3多不饱和脂肪酸作为细胞构成的重要成分,一直以来受到人们的广泛关注。研究表明,哺乳动物体内一定量的n-3多不饱和脂肪酸含量,可以起到预防心血管疾病、神经退行性疾病甚至癌症的作用。但n-3多不饱和脂肪酸在大多数动物体内不能合成,只能从食物中补充。fat-1基因的发现改变了这一现状,它可以将多不饱和脂肪酸从n-6形式转化为n-3形式,并发挥功能。研究人员已经利用转基因的方法制备了能够表达fat-1基因的转基因动物,如:小鼠、猪等,为研究n-3多不饱和脂肪酸的功能提供了高效、准确的医学、营养学动物模型。随着人们对n-3多不饱和脂肪酸认识的增加,对富含这一成分的食品需求也大大增加,fat-1基因在生产特殊农产品方面也具有十分大的潜力,亟待我们开发利用。本实验采用经密码子优化了的线虫基因sfat-1,使用脂质体转染的方法获得转sfat-1基因荷斯坦奶牛细胞,并将其应用到克隆研究中。实验中通过PCR-酶切连接方法人工合成了sfat-1基因,并构建了基于真核表达载体pCDNA3.1(+)的转基因载体pCDNA3.1(+)-sfat-1-EGFP及阴性对照载体pCDNA3.1(+)-EGFP;利用阳离子脂质体转染法,对自建8079TE细胞系进行转基因操作,经过G418筛选获得阳性转基因细胞,经PCR、RT-PCR检测证明外源基因已整合进入细胞基因组内。作为阶段性成果,本实验构建的pCDNA3.1(+)-EGFP对照载体为实验室后来转基因研究提供了良好的对照;建立的8079TE细胞系可以为多种基因的转基因克隆提供良好供体;筛选获得的转sfat-1基因细胞为后期n-3多不饱和脂肪酸细胞水平生物学研究及转sfat-1基因克隆牛研究奠定了基础。更多还原

【Abstract】 n-3 polyunsaturated fatty acids (PUFAs) that is an important component of the fatty acid, are concerned by scienists for a long time. It is necessary to maintain the appropriate proportion of n-3 PUFAs physiological and it can serve to prevent cardiovascular disease, neurodegenerative diseases and even cancer role. However most animals have to obtain n-3 PUFAs from diet, because they can not synthesize them by themselves. Fat-1 gene, translating into n-3 PUFA desaturase, can convert PUFAs from n-6 to n3 form. Researchers have used the method of gene transfer to the fat-1 gene expression in transgenic animals, such as: mice, pigs for the study of n-3 polyunsaturated fatty acids to provide the functions of an efficient and accurate medical, nutritional animal model. As the awareness of n-3 polyunsaturated fatty acids increasing,the rich ingredients of food demand also increased significantly in people. In the production of special agricultural products,fat-1 gene also has very great potential and we need development and utilization. The nematode gene sfat-1 which optimized the codon is used in this study, we use lipid-transfer method to obtain sfat-1 gene transfered Holstein cow cells, and apply it to cloning research.We synthetic sfat-1 gene using PCR-restriction method. Then reconstruct the gene vector pCDNA3.1(+)-sfat-1-EGFP and negative control vector pCDNA3.1(+)-EGFP based on the Construction of eukaryotic expression vector pCDNA3.1(+).The 8079 TE cell line is transferred using cationic lipid-transfer method, and the positive transgenic cells are obtained after G418 selection. After testing, the PCR, RT-PCR test proved that foreign genes has been integrated within the genome.As initial results, construction of the pCDNA3.1 (+)-EGFP vector is a good negative control in transgenic rearch in my lab.. The establishment of the 8079 TE cell lines can be good donors in variety of transgenic cloned research.Obtaining of sfat-1 gene transfected positive cells lays the foundation for research of n-3 polyunsaturated fatty acids biology at cell level and sfat-1 transgenic cattle.更多还原