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.