【作者】 刘晶；

【导师】 赵志辉；

【作者基本信息】 吉林大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 多不饱和脂肪酸(Polyunsaturated Fatty acids, PUFAs)参与构成生物膜,对生物膜的形成和物理性质、膜脂中脂肪酸的组成和不饱和度等方面起着重要的调节作用。人体中ω-3多不饱和脂肪酸含量低,而ω-6多不饱和脂肪酸含量相对偏高,且二者的平衡对于有机体自身的稳定和正常发育非常重要。除此之外,ω-3多不饱和脂肪酸对人类及其他哺乳动物的正常发育和维持健康极其重要,且在人类的多种疾病的预防与治疗中也发挥着突出的作用。但是ω-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。研究发现Fat-1基因编码的蛋白是多不饱和脂肪酸合成过程中一个重要的脂肪酸脱氢酶,即ω-3脂肪酸脱氢酶,它可以催化ω-6脂肪酸转化为ω-3脂肪酸。本实验首先根据Genebank上公布的秀丽隐杆线虫Fat-1基因序列,按照牛乳蛋白密码子的使用原则,对Fat-1基因进行了改造,同时在改造基因序列的5’端和3’端引入XhoI酶切位点,设计好的整个序列由上海生物工程技术服务有限公司合成。之后构建了基于乳腺特异性表达载体pBC1的转基因载体pBC1-Fat-1,采用原核显微注射方法获得了转Fat-1基因的转基因小鼠,经PCR、Southern blot检测证明外源基因已整合进入细胞基因组内,并对阳性母鼠奶样中ω-3多不饱和脂肪酸含量进行检测,用以分析Fat-1基因的表达情况。检测结果显示阳性母鼠奶样中ω-3/ω-6多不饱和脂肪酸的比值有所提高,说明此Fat-1基因可以在小鼠体内行使其功能,它的表达可以使小鼠乳中的ω-6多不饱和脂肪酸转化为ω-3多不饱和脂肪酸。更多还原

【Abstract】 Polyunsaturated fatty acids(PUFAs)are present in all groups of organisms and play a key role in the maintenance of the proper structure and functioning of biological membanes. The amount ofω-3 PUFAs is insufficient in most people, whereas the level ofω-6 PUFAs is relatively too high, the balance of these two classes of PUFAs is important for homeostasis and normal development. Besidesω-3 PUFAs have shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. However most animals have to obtainω-3 PUFAs from diet, because they can not synthesize them by themselves. The protein encoded by Fat-1 gene isω-3 fatty acid desaturase, which is an important fatty acid desaturase in the biosynthesis of PUFAs.This enzyme can catalyze the conversion ofω-6 PUFAs toω-3 PUFAs. In this study , based on the published seqence of the C.elegans Fat-1 gene in the Genebank and the regulation of the codons usage of the bovine milk protein with the bias, we optimized the codons of Fat-1 gene and synthesized the optimized gene, and inserted the restriction enzyme digestion sites XhoI to the 5’and 3’ends of sequende. After the whole modified sequence was synthesized by the Shanghai Sangon Biological Engineering Technology and Service Co,Ltd. Then we reconstruct the gene vector pBC1-Fat-1 based on the pBC1. After that we transferred this Fat-1 gene into mice by the method of microinjection. Then we proved that foreign genes has been integated within the mice genome by the PCR and Southern blot.Meanwhile we analysis theω-3/ω-6 PUFAs of the transgenic mice milk, the result demonstrated clearly thatω-6 PUFAs in milk of the transgenic mice are reduced,indicating thatω-6 PUFAs have been converted toω-3 PUFAs,causing the ratio ofω-3/ω-6 PUFAs to increased.更多还原