Lipids

, Volume 41, Issue 11, pp 1003–1016 | Cite as

Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional regulation of fatty acyl Δ6 desaturase of Atlantic cod (Gadus morhua L.)

  • Douglas R. Tocher
  • Xiaozhong Zheng
  • Christian Schlechtriem
  • Nicola Hastings
  • James R. Dick
  • Alan J. Teale
Articles

Abstract

This study reports the cloning, functional characterization, tissue expression, and nutritional regulation of a Δ6 fatty acyl desaturase of Atlantic cod (Gadus morhua). PCR primers were designed based on the sequences of conserved motifs in available fish desaturases and used to isolate a cDNA fragment from cod liver, with full-length cDNA obtained by rapid amplification of cDNA ends. The cDNA for the putative desaturase was shown to comprise 1980 bp, including a 261-bp 5′-UTR, a 375-bp 3′-UTR, and an ORF of 1344 bp that specified a protein of 447 amino acids. The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the heme-binding motif HPGG, all characteristic of microsomal fatty acyl desaturases. The cDNA displayed Δ6 desaturase activity in a yeast expression system. Quantitative real-time PCR assay of gene expression in cod showed that the Δ6 desaturase gene was expressed highly in brain, to a slightly lesser extent in liver, kidney, intestine, red muscle, and gill, and at much lower levels in white muscle, spleen, and heart. The expression of the Δ6 desaturase gene did not appear to be under significant nutritional regulation, with levels in liver and intestine being barely altered in fish fed a vegetable oil blend, in comparison with levels in fish fed fish oil. This was reflected in enzyme activity, as hepatocytes or enterocytes showed very little highly unsaturated FA biosynthesis activity irrespective of diet. Further studies are required to determine why the Δ6 desaturase appears to be barely functional in cod under the conditions tested.

Abbreviations

FO

fish oil

HUFA

highly unsaturated FA (carbon chain length≥C20 with≥3 double bonds)

ORF

open reading frame

Q-PCR

quantitative real-time polymerase chain reaction

RACE

rapid amplification of cDNA ends

SCMM-uracil

Saccharomyces cerevisiae minimal medium minus uracil

UPM

universal primer mix

UTR

untranslated region

VO

vegetable oil

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References

  1. 1.
    Simopoulos, A.P. (2000) Human Requirements for n−3 Polyunsaturated Fatty Acids, Poult. Sci. 79, 961–970.PubMedGoogle Scholar
  2. 2.
    Tidwell, J.H., and Allan, G.L. (2002) Fish as Food: Aquaculture’s Contribution, World Aquaculture 33, 44–48.Google Scholar
  3. 3.
    Tacon, A.G.J. (2004) Use of Fish Meal and Fish Oil in Aquaculture: A Global Perspective, Aquatic Resources, Culture and Development 1, 3–14.CrossRefGoogle Scholar
  4. 4.
    Pike, I. (2005) Eco-efficiency in Aquaculture: Global Catch of Wild Fish Used in Aquaculture, Internat. Aquafeed 8, 38–40.Google Scholar
  5. 5.
    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.Google Scholar
  6. 6.
    Tocher, D.R. (2003) Metabolism and Functions of Lipids and Fatty Acids in Teleost Fish, Rev. Fisheries Sci. 11, 107–184.Google Scholar
  7. 7.
    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.PubMedGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    Izquierdo, M., Obach, A., Arantzamendi, L., Montero, D., Robaina, L., and Rosenlund, G. (2003) Dietary Lipid Sources for Seabream and Seabass: Growth Performance, Tissue Composition and Flesh Quality, Aquacult. Nutr. 9, 397–407.CrossRefGoogle Scholar
  10. 10.
    Regost, C., Arzel, J., Robin, J., Rosenlund, G., and Kaushik, S.J. (2003) Total Replacement of Fish Oil by Soybean or Linseed Oil with a Return to Fish Oil in Turbot (Psetta maxima). 1. Growth Performance, Flesh Fatty Acid Profile, and Lipid Metabolism, Aquaculture 217, 465–482.CrossRefGoogle Scholar
  11. 11.
    Kurlansky, M. (1998) Cod: A Biography of the Fish That Changed the World, Johnathan Cape, London.Google Scholar
  12. 12.
    Brown, J.A., Minkoff, G., and Puvanendran, V. (2003) Larviculture of Atlantic Cod (Gadus morhua): Progress, Protocols and Problems, Aquaculture 227, 357–372.CrossRefGoogle Scholar
  13. 13.
    Morais, S., Bell, J.G., Robertson, D.A., Roy, W.J., and Morris, P.C. (2001) Protein/Lipid Ratios in Extruded Diets for Atlantic Cod (Gadus morhua L.): Effects on Growth, Feed Utilization, Muscle Composition and Liver Histology, Aquaculture 203, 101–119.CrossRefGoogle Scholar
  14. 14.
    Hemre, G.-I., Karlsen, O., Mangor-Jensen, A., and Rosenlund, G. (2003) Digestibility of Dry Matter, Protein, Starch and Lipid by Cod, Gadus morhua: Comparison of Sampling Methods, Aquaculture 225, 225–232.CrossRefGoogle Scholar
  15. 15.
    Hemre, G.-I., Karlsen, O., Eckhoff, K., Viet, K., Mangor-Jensen, A., and Rosenlund, G. (2004) Effect of Season, Light Regime and Diet on Muscle Composition and Selected Parameters in Farmed Atlantic Cod, Gadus morhua L., Aquacult. Res. 35, 683–697.CrossRefGoogle Scholar
  16. 16.
    Lall, S.P., and Nanton, D. (2002) Nutrition of Atlantic Cod, Bull. Aquacult. Assoc. Can. 102, 23–26.Google Scholar
  17. 17.
    Pavlov, D., Kjorsvik, E., Refsti, T., and Andersen, O. (2004) Broodstock and Egg Production, in Culture of Cold Water Marine Fish, Moksness, E., Kjorsvik, E., and Olsen, Y., eds., 129–203, Blackwell, Oxford.Google Scholar
  18. 18.
    Salze, G., Tocher, D.R., Roy, W.J., and Robertson, D.A. (2005) Egg Quality Determinants in Cod (Gadus morhua L.): Egg Performance and Lipids in Eggs from Farmed and Wild Broodstock, Aquacult. Res. 36, 1488–1499.CrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    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.Google Scholar
  21. 21.
    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. 6, 463–474.PubMedCrossRefGoogle Scholar
  22. 22.
    Zheng, X., Tocher, D.R., Dickson, C.A., Dick, J.R., Bell, J.G., and Teale, A.J. (2005) 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.PubMedCrossRefGoogle Scholar
  23. 23.
    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.Google Scholar
  24. 24.
    Zheng, X., Seiliez, I., Hastings, N., Tocher, D.R., Panserat, S., Dickson, C.A., Bergot, P., and Teale, A.J. (2004) Characterization and Comparison of Fatty Acyl Δ6 Desaturase cDNAs from Freshwater and Marine Teleost Fish Species, Comp. Biochem. Physiol. 139B, 269–279.Google Scholar
  25. 25.
    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.CrossRefGoogle Scholar
  26. 26.
    Zheng, X., Torstensen, B.E., Tocher, D.R., Dick, J.R., Henderson, R.J., and Bell, J.G. (2005) Environmental and Dietary Influences on Highly Unsaturated Fatty Acid Biosynthesis and Expression of Fatty Acyl Desaturase and Elongase Genes in Liver of Atlantic Salmon (Salmo salar), Biochim. Biophys. Acta 1734, 13–24.PubMedGoogle Scholar
  27. 27.
    U.S. National Research Council. (1993) Nutrient Requirements of Fish, National Academies Press, Washington, DC.Google Scholar
  28. 28.
    Agaba, M.K., Tocher, D.R., Dickson, C.A., Zheng, X., Dick, J.R., and Teale, A.J. (2005) Cloning and Functional Characterization of Polyunsaturated Fatty Acid Elongases from Marine and Freshwater Teleost Fish, Comp. Biochem. Physiol. 142B, 342–352.Google Scholar
  29. 29.
    Whelan, J.A., Russell, N.B., and Whelan, M.A. (2003) A Method for Absolute Quantification of cDNA Using Real-Time PCR, J. Immun. Method. 278, 261–269.CrossRefGoogle Scholar
  30. 30.
    Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497–509.PubMedGoogle Scholar
  31. 31.
    Christie, W.W. (2003) Preparation of Derivatives of Fatty Acids, in Lipid Analysis: Isolation, Separation and Structural Analysis of Lipids, vol. 15, pp. 205–225, J. Barnes and Associates, Bridgewater, UK.Google Scholar
  32. 32.
    Tocher, D.R., and Harvie, D.G. (1988) Fatty Acid Compositions of the Major Phosphoglycerides from Fish Neural Tissues: (n−3) and (n−6) Polyunsaturated Fatty Acids in Rainbow Trout (Salmo gairdneri, L.) and Cod (Gadus morhua) Brains and Retinas, Fish Physiol. Biochem. 5, 229–239.CrossRefGoogle Scholar
  33. 33.
    Ackman, R.G. (1980) Fish Lipids, Part 1, in Advances in Fish Science and Technology, Connell, J.J., ed., pp. 87–103, Fishing News Books, Farnham, UK.Google Scholar
  34. 34.
    Mourente, G., Dick, J.R., Bell, J.G., and Tocher, D.R. (2005) Effect of Partial Substitution of Dietary Fish Oil by Vegetable Oils on Desaturation and Oxidation of [1-14C]18∶3n−3 and [1-14C]20∶5n−3 in Hepatocytes and Enterocytes of European Sea Bass (Dicentrarchus labrax L.), Aquaculture 248, 173–186.CrossRefGoogle Scholar
  35. 35.
    Bell, J.G., Strachan, F., Good, J., and Tocher, D.R. (2006) Effect of Echium Oil on Growth, Fatty Acid Composition and Metabolism, Gill Prostaglandin Production and Macrophage Activity in Atlantic Cod (Gadus morhua L.), Aquaculture Res. 37, 606–617.CrossRefGoogle Scholar
  36. 36.
    Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem. 193, 265–275.PubMedGoogle Scholar
  37. 37.
    Ghioni, C., Tocher, D.R., and Sargent, J.R. (1997) The Effect of Culture on Morphology, Lipid and Fatty Acid Composition, and Polyunsaturated Fatty Acid Metabolism of Rainbow Trout (Oncorhynchus mykiss) Skin Cells, Fish Physiol. Biochem. 16, 499–513.CrossRefGoogle Scholar
  38. 38.
    Wilson, R., and Sargent, J.R. (1992) High Resolution Separation of Polyunsaturated Fatty Acids by Argentation Thin-Layer Chromatography, J. Chromatogr. 623, 403–407.CrossRefGoogle Scholar
  39. 39.
    Stubhaug, I., Tocher, D.R., Bell, J.G., Dick, J.R., and Torstensen, B.E. (2005) Fatty Acid Metabolism in Atlantic Salmon (Salmo salar L.) Hepatocytes, and Influence of Dietary Vegetable Oil, Biochim. Biophys. Acta 1734, 277–288.PubMedGoogle Scholar
  40. 40.
    Saitou, N., and Nei, M. (1987) The Neighbor-Joining Method. A New Method for Reconstructing Phylogenetic Trees, Mol. Biol. Evol. 4, 406–425.PubMedGoogle Scholar
  41. 41.
    Zar, J.H. (1984) Biostatistical Analysis, 2nd edn., Prentice-Hall, Englewood Cliffs, NJ.Google Scholar
  42. 42.
    Nelson, J.S. (1994) Fishes of the World, 3rd edn., John Wiley and Sons, New York.Google Scholar
  43. 43.
    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.CrossRefGoogle Scholar
  44. 44.
    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 39, 39–44.CrossRefGoogle Scholar
  45. 45.
    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.PubMedCrossRefGoogle Scholar
  46. 46.
    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.PubMedCrossRefGoogle Scholar
  47. 47.
    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.PubMedCrossRefGoogle Scholar
  48. 48.
    Leonard, A.E., Bobik, E.G., Dorado, J., Kroeger, P.E., Chuang, L.-T., Thurmond, J.M., Parker-Barnes, J.M., Das, T., Huang, Y.-S., and Murkerji, P. (2000) Cloning of a Human cDNA Encoding a Novel Enzyme Involved in the Elongation of Long Chain Polyunsaturated Fatty Acids, Biochem. J. 350, 765–770.PubMedCrossRefGoogle Scholar
  49. 49.
    Leonard, A.E., Kelder, B., Bobik, E.G., Chuang, L.-T., Lewis, C.J., Kopchick, J.J., Murkerji, P., and Huang, Y.-S. (2002) Identification and Expression of Mammalian Long-Chain PUFA Elongation Enzymes, Lipids 37, 733–740.PubMedCrossRefGoogle Scholar
  50. 50.
    Inagaki, K., Aki, T., Fukuda, Y., Kawamoto, S., Shigeta, S., Ono, K. and Suzuki, O. (2002) Identification and Expression of a Rat Fatty Acid Elongase Involved the Biosynthesis of C18 Fatty Acids, Biosci. Biotechnol. Biochem. 66, 613–621.PubMedCrossRefGoogle Scholar

Copyright information

© AOCS Press 2006

Authors and Affiliations

  • Douglas R. Tocher
    • 1
  • Xiaozhong Zheng
    • 1
  • Christian Schlechtriem
    • 1
  • Nicola Hastings
    • 1
  • James R. Dick
    • 1
  • Alan J. Teale
    • 1
  1. 1.Institute of AquacultureUniversity of StirlingStirlingScotland, UK

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