人泛素启动子UbB介导Fat-1基因在绵羊成纤维细胞内的表达

畜牧兽医学报

人泛素启动子UbB介导Fat-1基因在绵羊成纤维细胞内的表达

毛志福^1,2，王立民²，张银国^1*，周平^2*

1.石河子大学动物科技学院，石河子 832000;2.新疆农垦科学院，新疆兵团绵羊繁育生物技术重点实验室，石河子 832000

收稿日期:2012-11-21 出版日期:2013-04-23 发布日期:2013-04-23
通讯作者: 张银国（1956-），男，教授，硕士生导师，主要从事动物疾病诊疗与繁殖研究，E-mail：zyg@shzu.edu.cn；周平（1974-），副研究员，硕士生导师，主要从事动物繁殖育种研究，E-mail：zhpxqf@163.com
作者简介:毛志福(1985 - ),男，甘肃武威人，硕士生，主要从事动物临床疾病方面研究，E-mail：maozify@sina.com
基金资助:
转Fat-1基因高品质肉羊新品种的培育（2009ZX08008-008B）；脂肪酸脱氢酶1（FAD1）转基因羊的研究(2011BB014)

Expression of Unsaturated Fatty Acid Dehydrogenase Gene in Sheep Fibroblast Driven by Human Ubiquitin Promoter UbB

MAO Zhi-fu^1,2, WANG Li-min², ZHANG Yin-guo^1*, ZHOU Ping^2*

1. College of Animal Science & Technology Shihezi University, Shihezi 832000, China;2. The Breed & Biological Key Laboratory of Sheep, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China

Received:2012-11-21 Online:2013-04-23 Published:2013-04-23

摘要/Abstract

摘要：

旨在构建不饱和脂肪酸脱氢酶基因Fat-1表达载体，实现人泛素启动子UbB调控外源基因Fat-1在绵羊细胞内表达。PCR扩增得到UbB序列，秀丽隐杆线虫Fat-1基因密码子根据绵羊对同义密码子使用偏好性优化后，由公司合成获得oFat-1基因序列，以及pcDNA3.1中的GBHpolyA序列共同插入pCMV-DsRed2-1载体的多克隆位点（MCS），得到重组真核表达载体pUbB-oFat-polyA-CMV-DsRed，载体序列经酶切测序鉴定。线性化的表达载体采用脂质体包裹后转染绵羊成纤维细胞，经G418抗性和红色荧光标记筛选得到阳性细胞，并对阳性细胞进行PCR和反转录PCR鉴定，HPLC检测细胞中不饱和脂肪酸的变化。构建的真核表达载体序列正确，Fat-1基因能够在绵羊成纤维细胞基因组中正常表达，促进细胞ω-6PUFAs向ω-3PUFAs转化，ω-3PUFAs占总量的百分比从11.48%增加到24.41%,ω-6PUFAs的百分比从88.52%下降到75.59%，ω-6PUFAs/ω-3PUFAs比值从7.82±0.18下降到3.10±0.03，差异极显著（P＜0.01）。成功构建了UbB启动子介导的Fat-1真核表达载体,筛选得到表达不饱和脂肪酸脱氢酶的绵羊细胞，为进一步制备转基因克隆绵羊奠定了基础。

Abstract:

The aim of this study was to construct expression vector of Fat-1 in sheep fibroblasts,encoding unsaturated fatty acid desaturase driven by UbB(human sapiens Ubiquitin gen B sufamily) constitutive promoter. UbB was obtained by PCR, Fat-1 gene from Caenorhabdit elegans was optimized base on ovis aries codon，and GBHpolyA was from pcDNA3.1.These donor sequences were orderly inserted into MCS of pCMV-DsRed2-1 vector. The recombinant vector(pUbB-oFat-polyA-CMV-DsRed) was analysed by endonucleases digestion and sequencing. The linearized vector was transfected into sheep fibroblast with liposome. The positive cell was selected with G418 and red fluorescence signal,and furtherly confirmed by PCR and RT-PCR,components of PUFAs were detected by HPLC. The results showed that corrcet recombinant vector was obtained,and Fat-1 gene could been properly expressed in sheep fibroblast. The ω-3 PUFAs were greatly increased from 11.48% to 24.41% and simultaneously the ω-6 PUFAs decreased from 88.52% to 75.59%.A significantly reduction of ω-6/ω-3PUFAs ratio was observed from 7.82±0.18 to 3.10±0.03(P＜0.01). These results demonstrate that the expression vector was constructed successfully, and the sheep fibroblast line was obtained, which could encode unsaturated fatty acid desaturase, furthermore, this study may pave the way to generate transgenic sheep by somatic cell nuclear transfer technology.

中图分类号:

S826.8.2

毛志福，王立民，张银国，周平. 人泛素启动子UbB介导Fat-1基因在绵羊成纤维细胞内的表达[J]. 畜牧兽医学报, doi: .

MAO Zhi-fu, WANG Li-min, ZHANG Yin-guo, ZHOU Ping. Expression of Unsaturated Fatty Acid Dehydrogenase Gene in Sheep Fibroblast Driven by Human Ubiquitin Promoter UbB[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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