Cloning, Expression and Functional Studies of Omega-3 Fatty Acid Desaturase Gene

Abstract: Omega-3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and play a particularly important role in pharmaceutical and nutraceutical fields. The amount ofω-3 PUFAs is insufficient in most people, whereas the level ofω-6 PUFAs is relatively too high, with anω-6/ω-3 ratio of >10. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations ofω-3 PUFAs in mammals relies on chronic dietary intake of fat rich inω-3 PUFAs, because human lack enzymatic activities necessary either to synthesize the precursor ofω-3 PUFAs or to convertω-6 toω-3 PUFAs. Although dietary supplementation withω-3 PUFAs is a safe intervention, it has a number of limitations.In this study, a gene named fat-1 in Caenorhabditis elegans was cloned by RT-PCR at first, 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 by introducing anω-3 double bond into their hydrocarbon chains.The sequences of fat-1 gene N-terminal were cloned into fusion expression vector pGEX-4T-2 to generate recombinant plasmid pGEX-fat1-N. After expressing in Escherichia coli, the expressed product GST-fat1-N fusion protein was purified and used to immunize rabbit. The antibody against GST-fat1-N protein has a good affinity and selectivity by enzyme-linked immunosorbent assay.Two kinds of expression vector pfat1-GFP and pfat-1 were constructed, purified plasmids used to transfect human embryonic kidney (HEK293T) cells. The result demonstrated clearly that heterologous expression of the fat-1 gene in HEK293T rendered cells capable o…
Key words: Caenorhabditis elegans; ω-3 fatty acid desaturase; fat-1 gene; gene cloning; expression

This entry was posted in Master Thesis. Bookmark the permalink.