The Expression Of Fat-1 Gene In Fiber Cells From Rabbits Fetus

ω-3 polyunsaturated fatty acids (co-3 PUFAS) is one of the essential fatty acids in human. Previous researches reported that when theω-6 toω-3 PUFAS ratio was 18, it would significantly increase the risks for breast cancer, colorectal cancer and prostate cancer. But there is no fat-1 gene which can codeω-3 PUFAS in mammal. In order to change the content ofω-3 PUFAS in mammals, modern transgenic technology makes it possible. In this experiment, we chose the Belgium rabbits embryo of 15 to 17 gestational age and the small Caenorhabditis elegans as the experimental materials, to study the expression of fat-1 gene in rabbits, which has important theoretical and practical significance to nurture transgenic animals withω-3 PUFAS and establish Animal model.The experiments cultured rabbit fibroblasts successfully through tissue culture method.According to previous report by GenBank on the sequence of CDS region of Caenorhabditis elegans fat-1 gene, primer was designed to clone it through RT-PCR. The gained elegans fat-1 gene fragments, which were sub-cloned into pMD19-T vectors to produce intermediate vector pMD19-T-fat-1, were confirmed through sequencing comparison to be in full accord with the reference sequence fat-1 gene by GenBank. The forecast and bioinformatics analysis on nucleotide sequence comparison, properties and structures of coding proteins of fat-1 gene, showed that the sequence of CDS region of fat-1 gene has a whole length of 1209 bp and code 402 amino acids; the coding protein (46.5KD molecular weight and 6.14 isoelectric point)belonging to trans-membrane protein superfamily, owns a FADS domain whose secondary structure is on the basis of two a helical-structure between 72-123 and 232-281 amino acid sites.Vector pMD19-T-fat-1 and eukaryotic expression vector pEGFP-N1 were enzyme digested at suitable sites, as well recombinant vector pfat-1-GFP was obtained through fat-1 gene fragment joined to plasmid vector framework pEGFP-N1.48h after recombinant plasmids transfecting rabbit fibroblasts, the cells fluoresce under fluorescence inverted microscope. Supernatants were analyzed by Westernblot and gained a 47KD stripe which fitted well with the 46.5KD theoretical value of the product translated by fat-1 gene, which demonstrated that the fat-1 gene had expressed in rabbit fibroblasts.