Abstract
Fish are an important source of the n−3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a Δ5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterization of a Δ6 fatty acyl desaturase of Atlantic salmon and describe its genomic structure, tissue expression, and nutritional regulation. A salmon genomic library was screened with a salmon Δ5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106 bp containing an open reading frame of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly Δ6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the Δ6 desaturase gene constituted 13 exons in 7965 bp of genomic DNA. Quantitative real-time PCR assay of gene expression in Atlantic salmon showed that both Δ6 and Δ5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver, and brain, and less so in kidney, heart, gill, adipose tissue, muscle, and spleen. Furthermore, expression of both Δ6 and Δ5 fatty acyl desaturase genes in intestine, liver, red muscle, and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- FO:
-
fish oil
- HUFA:
-
highly unsaturated fatty acids (carbon chain length ≥20 with ≥3 double bonds)
- MMLV:
-
Moloney murine leukemia virus
- ORF:
-
open reading frame
- qrtPCR:
-
quantitative (real-time) polymerase chain reaction
- RACE:
-
rapid amplification of cDNA ends
- SCMM:
-
Saccharomyces cerevisiae minimal medium
- UTR:
-
untranslated region
- VO:
-
vegetable oil
References
Simopoulos, A.P. (1989) Summary of the NATO Advanced Research Workshop on Dietary Omega 3 and Omega 6 Fatty Acids: Biological Effects and Nutritional Essentiality, J. Nutr. 119, 521–528.
Simopoulos, A.P. (1991) Omega-3 Fatty Acids in Health and Disease and in Growth and Development, Am. J. Clin. Nutr. 54, 438–463.
Lands, W.E. (1992) Biochemistry and Physiology of n−3 Fatty Acids, FASEB J. 6, 2530–2536.
Tidwell, J.H., and Allan, G.L. (2002) Fish as Food: Aquaculture's Contribution, World Aquaculture 33, 44–48.
Sargent, J.R., and Tacon, A. (1999) Development of Farmed Fish: A Nutritionally Necessary Alternative to Meat, Proc. Nutr. Soc. 58, 377–383.
Barlow, S. (2000) Fish Meal and Fish Oil: Sustainable Feed Ingredients for Aquafeeds, Global Aquacult. Advocate 4, 85–88.
Sargent, J.R., Tocher, D.R., and Bell, J.G. (2002) The Lipids, in Fish Nutrition, 3rd edn. (Halver, J.E., and Hardy, R.W., eds.), pp. 181–257, Academic Press, San Diego.
Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., and Sargent, J.R. (2001) Replacement of Fish Oil with Rape Seed Oil in Diets of Atlantic Salmon (Salmos salar) Affects Tissue Lipid Compositions and Hepatocyte Fatty Acid Metabolism, J. Nutr. 131, 1535–1543.
Bell, J.G., Tocher, D.R., Farndale, B.M., Cox, D.I., McKinney, R.W., and Sargent, J.R. (1997) The Effect of Dietary Lipid on Polyunsaturated Fatty Acid Metabolism in Atlantic Salmon (Salmo salar) Undergoing Parr-Smolt Transformation, Lipids 32, 515–525.
Tocher, D.R., Bell, J.G., Henderson, R.J., McGhee, F., Mitchell, D., and Morris, P.C. (2000) The Effect of Dietary Linseed and Rapeseed Oils on Polyunsaturated Fatty Acid Metabolism in Atlantic Salmon (Salmo salar) Undergoing Parr-Smolt Transformation, Fish Physiol. Biochem. 23, 59–73.
Tocher, D.R., Bell, J.G., Dick, J.R., and Crampton, V.O. (2003) Effects of Vegetable Oil Diets on Atlantic Salmon Hepatocyte Desaturase Activities and Liver Fatty Acid Compositions, Lipids 38, 723–732.
Tocher, D.R., Leaver, M.J., and Hodgson, P.A. (1998). Recent Advances in the Biochemistry and Molecular Biology of Fatty Acyl Desaturases, Prog. Lipid Res. 37, 73–117.
Huang, Y.-S., Chaudhary, S., Thurmond, J., Bobik, E.G., Yuan, L., Chan, G.E., Kirchner, S.J., Mukerji, P., and Knutson, D.S. (1999) Cloning of Δ12- and Δ5-Desaturases from Mortierella alpina and Recombinant Production of γ-Linolenic Acid in Saccharomyces cerevisiae, Lipids 34, 649–659.
Napier, J.A., Hey, S.J., Lacey, D.J., and Shewry, P.R. (1998) Identification of a Caenorhabditis elegans Δ6-Fatty-Acid-Desaturase by Heterologous Expression in Saccharomyces cerevisiae, Biochem. J. 330, 611–614.
Aki, T., Shimada, Y., Inagaki, K., Higashimoto, H., Kawamoto, S., Shiget, S., Ono, K., and Suzuki, O. (1999) Molecular Cloning and Functional Characterization of Rat Δ-6 Fatty Acid Desaturase, Biochem. Biophys. Res. Commun. 255, 575–579.
Cho, H.P., Nakamura, M.T., and Clarke, S.D. (1999) Cloning, Expression and Nutritional Regulation of the Human Δ6 Desaturase, J. Biol. Chem. 274, 471–477.
Michaelson, L.V., Lazarus, C.M., Griffiths, G., Napier, J.A., and Stobart, A.K. (1998) Isolation of a Δ5 Fatty Acid Desaturase Gene from Mortierella alpina, J. Biol. Chem. 273, 19055–19059.
Michaelson, L.V., Napier, J.A., Lewis, M., Griffiths, G., Lazarus, C.M., and Stobart, A.K. (1998) Functional Identification of a Fatty Acid Δ5 Desaturase Gene from Caenorhabditis elegans, FEBS Lett. 439, 215–218.
Watts, J.L., and Browse, J. (1999) Isolation and Characterization of a Δ5 Fatty Acid Desaturase Fatty Acid Desaturase from Caenorhabditis elegans, Arch. Biochem. Biophys. 362, 175–182.
Cho, H.P., Nakamura, M.T., and Clarke, S.D. (1999) Cloning, Expression and Nutritional Regulation of the Human Δ5 Desaturase, J. Biol. Chem. 274, 37335–37339.
Leonard, A.E., Kelder, B., Bobik, E.G., Chuang, L.-T., Parker-Barnes, J.M., Thurmond, J.M., Kroeger, P.E., Kopchick, J.J., Huang, Y.-S., and Mukerji, P. (2000) cDNA Cloning and Characterization of Human Δ5 Desaturase Involved in the Biosynthesis of Arachidonic Acid, Biochem. J. 347, 719–724.
Hastings, N., Agaba, M., Tocher, D.R., Leaver, M.J., Dick, J.R., Sargent, J.R., and Teale, A.J. (2001) A Vertebrate Fatty Acid Desaturase with Δ5 and Δ6 Activities, Proc. Natl. Acad. Sci. USA 98, 14304–14309.
Seiliez, I., Panserat, S., Kaushik, S., and Bergot, P. (2001) Cloning, Tissue Distribution and Nutritional Regulation of a Δ6-Desaturase-like Enzyme in Rainbow Trout, Comp. Biochem. Physiol. 130B, 83–93.
Seiliez, I., Panserat, S., Corraze, G., Kaushik, S., and Bergot, P. (2003) Cloning and Nutritional Regulation of a Δ6-Desaturase-like Enzyme in the Marine Teleost Gilthead Seabream (Sparus aurata), Comp. Biochem. Physiol. 135B, 449–460.
Zheng, X., Seiliez, I., Hastings, N., Tocher, D.R., Panserat, S., Dickson, C.A., Bergot, P., and Teale A.J. (2004) Characterisation and Comparison of Fatty Acyl Δ6 Desaturase cDNAs from Freshwater and Marine Teleost Fish Species, Comp. Biochem. Physiol. 139B, 269–279.
Hastings, N., Agaba, M.K., Tocher, D.R., Zheng, X., Dickson, C.A., Dick, J.R., and Teale, A.J. (2004) Molecular Cloning and Functional Characterization of Fatty Acyl Desaturase and Elongase cDNAs Involved in the Production of Eicosapentaenoic and Docosahexaenoic Acids from α-Linolenic Acid in Atlantic Salmon (Salmo salar), Mar. Biotechnol. in press.
U.S. National Research Council (1993) Nutrient Requirements of Fish, National Academy Press, Washington, DC.
Saitou, N., and Nei, M. (1987) The Neighbor-Joining Method. A New Method for Reconstructing Phylogenetic Trees, Mol. Biol. Evol. 4, 406–425.
Napier, J.A., Michaelson, L.V., and Sayanova, O. (2003) The Role of Cytochrome b 5 Fusion Desaturases in the Synthesis of Polyunsaturated Fatty Acids, Prostaglandins Leukotrienes Essent. Fatty Acids 68, 135–143.
Tocher, D.R., and Sargent, J.R. (1990) Incorporation into Phospholipid Classes and Metabolism via Desaturation and Elongation of Various 14C-Labelled (n−3) and (n−6) Polyunsaturated Fatty Acids in Trout Astrocytes in Primary Culture, J. Neurochem. 54, 2118–2124.
Tocher, D.R., and Sargent, J.R. (1990) Effect of Temperature on the Incorporation into Phospholipid Classes and the Metabolism via Desaturation and Elongation of (n−3) and (n−6) Polyunsaturated Fatty Acids in Fish Cells in Culture, Lipids 25, 435–442.
Sayanova, O.V., Beaudoin, F., Michaelson, L.V., Shewry, P.R., and Napier, J.A. (2003) Identification of Primula Fatty Acid Δ6-Desaturases with n−3 Substrate Preferences, FEBS Lett. 542, 100–104.
De Antueno, R.J., Knickle, L.C., Smith, H., Elliot, M.L., Allen, S.J., Nwaka, S., and Winther, M.D. (2001) Activity of Human Δ5 and Δ6 Desaturases on Multiple n−3 and n−6 Polyunsaturated Fatty Acids, FEBS Lett. 509, 77–80.
Allendorf, F.W., and Thorgaard, G.H. (1984) Tetraploidy and the Evolution of Salmonid Fishes, in Evolutionary Genetics of Fishes: Monographs in Evolutionary Biology (Turner, B.J., ed.), pp. 1–53, Plenum Press, New York.
Tocher, D.R. (2003) Metabolism and Functions of Lipids and Fatty Acids in Teleost Fish, Rev. Fisheries Sci. 11, 107–184.
Tocher, D.R., and Ghioni, C. (1999) Fatty Acid Metabolism in Marine Fish: Low Activity of Δ5 Desaturation in Gilthead Sea Bream (Sparus aurata) Cells, Lipids 34, 433–440.
Ghioni, C., Tocher, D.R., Bell, M.V., Dick, J.R., and Sargent, J.R. (1999) Low C18 to C20 Fatty Acid Elongase Activity and Limited Conversion of Stearidonic Acid, 18∶4n−3, to Eicosapentaenoic Acid, 20∶5n−3, in a Cell Line from the Turbot, Scophthalmus maximus, Biochim. Biophys. Acta 1437, 170–181.
Pereira, S.L., Leonard, A.E., and Mukerji, P. (2003) Recent Advances in the Study of Fatty Acid Desaturases from Animals and Lower Eukaryotes, Prostaglandins Leukotrienes Essent. Fatty Acids 68, 97–106.
Wallis, J.G., Watts, J.L., and Browse, J. (2002) Polyunsaturated Fatty Acid Synthesis: What Will They Think of Next?, Trends Biochem. Sci. 27, 467–473.
D'Andrea, S., Guillou, H., Jan, S., Catheline, D., Thibault, J.-N., Bouriel, M., Rioux, V., and Legrand, P. (2002) The Same Rat Δ6-Desaturase Not Only Acts on 18- but Also on 24-Carbon Fatty Acids in Very-Long-Chain Polyunsaturated Fatty Acid Biosynthesis, Biochem. J. 364, 49–55.
Tocher, D.R., Agaba, M., Hastings, N., and Teale, A.J. (2003) Biochemical and Molecular Studies of the Fatty Acid Desaturation Pathway in Fish, in The Big Fish Bang—Proceedings of the 26th Annual Larval Fish Conference (Browman, H.I., and Skiftesvik, A.B., eds.), pp. 211–227, Institute of Marine Nutrition, Bergen.
Qui, X., Hong, H., and MacKenzie, S.L. (2001) Identification of a Δ4 Fatty Acid Desaturase from Thraustochytrium sp. Involved in the Synthesis of Docosahexaenoic Acid by Heterologous Expression in Saccharomyces cerevisiae and Brassica juncea, J. Biol. Chem. 276, 31561–31566.
Meyer, A., Cirpus, P., Ott, C., Scheckler, R., Zahringer, U., and Heinz, E. (2003) Biosynthesis of Docosahexaenoic Acid in Euglena gracilis: Biochemical and Molecular Evidence for the Involvement of a Δ4-Fatty Acyl Group Desaturase, Biochemistry 42, 9779–9788.
Tonon, T., Harvey, D., Larson, T.R., and Graham, I.A. (2003) Identification of a Very Long Chain Polyunsaturated Fatty Acid Δ4-Desaturase from the Microalga Pavlova lutheri, FEBS Lett. 553, 440–444.
Marquardt, A., Stöhr, H., White, K., and Weber, B.H.F. (2000) cDNA Cloning, Genomic Structure, and Chromosomal Localization of Three Members of the Human Fatty Acid Desaturase Family, Genomics 66, 175–183.
Sperling, P., Ternes, P., Zank, T.K., and Heinz, E. (2003) The Evolution of Desaturases, Prostaglandins Leukotrienes Essent. Fatty Acids 68, 73–95.
Nelson, J.S. (1994) Fishes of the World, 3rd edn., John Wiley & Sons, New York.
Tocher, D.R., Fonseca-Madrigal, J., Bell, J.G., Dick, J.R., Henderson, R.J., and Sargent, J.R. (2002) Effects of Diets Containing Linseed Oil on Fatty Acid Desaturation and Oxidation in Hepatocytes and Intestinal Enterocytes in Atlantic Salmon (Salmo salar), Fish Physiol. Biochem. 26, 157–170.
Tocher, D.R., Fonseca-Madrigal, J., Dick, J.R., Ng, W.-K., Bell, J.G., and Campbell, P.J. (2004) Effects of Diets Containing Palm Oil and Water Temperature on Fatty Acid Desaturation and Oxidation in Hepatocytes and Intestinal Enterocytes in Rainbow Trout (Oncorhynchus mykiss), Comp. Biochem. Physiol. 137B, 49–63.
Bell, M.V., Dick, J.R., and Porter A.E.A. (2001) Biosynthesis and Tissue Deposition of Docosahexaenoic Acid (22∶6n−3) in Rainbow Trout (Oncorhynchus mykiss), Lipids 36, 1153–1159.
Bell, M.V., Dick, J.R., and Porter, A.E.A. (2003) Pyloric Ceca Are a Major Site of 22∶6n−3 Synthesis in Rainbow Trout (Oncorhynchus mykiss), Lipids 38, 39–44.
Brenner, R.R. (1989) Factors Influencing Fatty Acid Chain Elongation and Desaturation, in The Role of Fats in Human Nutrition (Vergrosesen, A.J., and Crawford, M., eds.), 2nd edn., pp. 45–79, Academic Press, San Diego.
Brenner, R.R. (1981) Nutritional and Hormonal Factors Influencing Desaturation of Essential Fatty Acids, Prog. Lipid Res. 20, 41–47.
Zheng, X., Tocher, D.R., Dickson, C.A., Bell, J.G., and Teale, A.J. (2004) Effects of Diets Containing Vegetable Oil on Expression of Genes Involved in Polyunsaturated Fatty Acid Biosynthesis in Liver of Atlantic Salmon (Salmo salar), Aquaculture 236, 467–483.
Author information
Authors and Affiliations
Corresponding author
About this article
Cite this article
Zheng, X., Tocher, D.R., Dickson, C.A. et al. Highly unsaturated fatty acid synthesis in vertebrates: New insights with the cloning and characterization of a Δ6 desaturase of Atlantic salmon. Lipids 40, 13–24 (2005). https://doi.org/10.1007/s11745-005-1355-7
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11745-005-1355-7