单 位：
西北农林科技大学动物医学院农业部动物生殖生理和胚胎工程重点开放实验室
关键词：
ω-3脂肪酸脱氢酶;不饱和脂肪酸;真核表达载体;牛胎儿成纤维细胞
摘 要：
培养线虫(Caenorhabditis elegans),提取RNA并通过PT-PCR获得其ω-3脂肪酸脱氢酶基因Fat-1,整合入真核表达载体,构建了哺乳动物细胞表达载体pFat-IRES2-GFP,转染牛胎儿成纤维细胞系并对其进行G418筛选获得稳定转染细胞株。对稳定转染Fat-1细胞株的RT-PCR分析及脂肪酸组成的HPLC分析结果表明,Fat-1基因已经整合入牛基因组中,并能够表达和发挥其ω-3脂肪酸脱氢酶的作用,促使-ω6PUFAs转变为相应的-ω3 PUFAs。ω-6脂肪酸总量从35.60%下降到25.47%,-ω3 PUFAs总量从5.75%上升到12.33%,多不饱和脂肪酸的比值从正常细胞中的6.19下降到阳性细胞中的2.07,说明Fat-1基因整合是成功的,能够在细胞中以相应的-ω6 PUFAs为底物合成-ω3 PUFAs。
译 名：
Construction and Expression of Mammal Expression Vector of ω-3 Fatty Acid Desaturase Gene Fat-1
作 者：
ZHAN Liping,LIU Jun,ZHENG Congying,ZHANG Bowei and ZHANG Yong(Key Laboratory of Animal Reproductive Physiology & Embryo Technology,Ministry of Agriculture,Northwest A&F University,Yangling Shaanxi 712100,China)
关键词：
ω-3 fatty acid desaturase gene;Polyunsaturated fatty acids;Mammal expression vector;Cattle fibroblasts of embryo
摘 要：
The Fat-1 gene was cloned by RT-PCR and constructed into a mammal expression vector in this study.The vector pFat-IRES2-GFP was introduced into cattle fibroblasts of embryo,and its expression elevated the cellularω-3 PUFAs contents quickly and effectively and improved the ratio of ω-6/ω-3 PUFAs.Cellular lipids extracts from stably selected cells were analyzed with HPLC and the results showed that amount of total ω-6 PUFAs dropped form 35.60% to 25.47%,whereas the amount of totalω-3 PUFAs increased form 5.75% to 12.33%.The ω-6/ω-3 ratio also dropped from 6.19 to 2.07.These data demonstrates the Fat-1 gene can produce ω-3 PUFAs by using the corresponding ω-6 PUFAs as substrates,which shows its potential use in the production of ω-3 PUFAs in transgenic animals.
