Skip to main content
SpringerLink
Log in

Cloning and molecular characterization of the Δ6-desaturase from two Echium plant species: Production of GLA by heterologous expression in yeast and tobacco

  • Articles
  • Published:
Lipids

Abstract

The synthesis of GLA (Δ6, 9, 12-18:3) is carried out in a number of plant taxa by introducing a double bond at the Δ6 position of its precursor, linoleic acid (Δ9, 12-18:2), through a reaction catalyzed by a Δ6-desaturase enzyme. We have cloned genes encoding the Δ6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b5 domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other Δ6-desaturases in a different cluster from that of the highly related Δ8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ALA:

α-linolenic acid

CaMV:

cauliflower mosaic virus

DIG:

digoxigenin

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

IPCR:

inverse polymerase chain reaction

LA:

Imoleic acid

MS:

mass spectrometry

OTA:

octadecatetraenoic acid

PCR:

polymerase chain reaction

References

  1. Gunstone, F.D. (1992) γ-Linolenic Acid-Occurrence and Physical and Chemical Properties, Prog. Lipid Res. 31, 145–161.

    Article  PubMed  CAS  Google Scholar 

  2. Gunstone, F.D. (1998) Movements Towards Tailor-Made Fats, Prog. Lipid Res. 37, 277–305.

    Article  PubMed  CAS  Google Scholar 

  3. Horrobin, D.F. (1992) Nutritional and Medical Importance of γ-Linolenic Acid, Prog. Lipid Res. 31, 163–194.

    Article  PubMed  CAS  Google Scholar 

  4. Brosche, T., and Platt, D. (2000) Effect of Borage Oil Consumption on Fatty Acid Metabolism, Transepidermal Water Loss and Skin Parameters in Elderly People, Arch. Gerontol. Geriatr. 30, 139–150.

    Article  PubMed  CAS  Google Scholar 

  5. Takahashi, Y., Ide, T., and Fujita, H. (2000) Dietary γ-Linolenic Acid in the Form of Borage Oil Causes Less Body Fat Accumulation Accompanying an Increase in Uncoupling Protein 1 mRNA Level in Brown Adipose Tissue, Comp. Biochem. Physiol. 127, 213–222.

    Article  CAS  Google Scholar 

  6. Das, U.N., Prasad, V.V.S.K., and Reddy, D.R. (1995) Local Application of γ-Linolenic Acid in the Treatment of Human Gliomas, Cancer Lett. 94, 147–155.

    Article  PubMed  CAS  Google Scholar 

  7. Preuss, M., Girnun, G.D., Darby, C.J., Khoo, N., Spector, A.A., and Robbins, M.E. (2000) Role of Antioxidant Enzyme Expression in the Selective Cyototoxic Response of Glioma Cell to γ-Linolenic Acid Supplementation, Free Radic. Biol. Med. 28, 1143–1156.

    Article  PubMed  CAS  Google Scholar 

  8. Harwood, J.L. (1996) Recent Advances in the Biosynthesis of Plant Fatty Acids, Biochim. Biophys. Acta 1301, 7–56.

    PubMed  Google Scholar 

  9. Griffiths, G., Brechany, E.Y., Jackson, F.M., Christie, W.W., Stymne, S., and Stobart, K. (1996) Distribution and Biosynthesis of Stearidonic Acid in Leaves of Borago officinalis. Phytochemistry 43, 381–386.

    Article  CAS  Google Scholar 

  10. Gill, I., and Valivety, R. (1997) Polyunsaturated Fatty Acids, Part 1: Occurrence, Biological Activities and Applications, Trends Biotechnol. 15, 401–409.

    Article  PubMed  CAS  Google Scholar 

  11. Napier, J.A., Michaelson, L.V., and Stobart, A.K. (1999) Plant Desaturases: Harvesting the Fat of the Land, Curr. Opin. Plant Biol. 2, 123–127.

    Article  PubMed  CAS  Google Scholar 

  12. Sakuradani, E., Kobayashi, M., and Shimizu, S. (1999) Δ6-Fatty Acid Desaturase From an Arachidonic Acid-Producing Mortierella Fungus. Gene Cloning and Its Heterologous Expression in a Fungus, Aspergillus, Gene 238, 445–453.

    Article  PubMed  CAS  Google Scholar 

  13. Huang, Y.-S., Chaudhary, S., Thurmond, J.M., Bobik, E.G., Yuan, L., Chan, G.M., Kirchner, S.J., Mukerji, P., and Knutzon, D.S. (1999) Cloning of Δ12- and Δ6-Desaturases from Mortierella alpina and Recombinant Production of γ-Linolenic Acid in Saccharomyces cerevisiae, Lipids 34, 649–659.

    Article  PubMed  CAS  Google Scholar 

  14. Murphy, D.J. (1995) The Use of Conventional and Molecular Genetics to Produce New Diversity in Seed Oil Composition for the Use of Plant Breeders—Progress, Problems and Future Prospects, Euphytica 85, 433–440.

    Article  CAS  Google Scholar 

  15. Reddy, A.S., and Thomas, T.L. (1996) Expression af a Cyanobacterial Δ6-Desaturase Gene Results in γ-Linolenic Acid Production in Transgenic Plants, Nat. Biotechnol. 14, 639–642.

    Article  PubMed  CAS  Google Scholar 

  16. López Alonso, D., and García Maroto, F. (2000) Plants as ‘Chemical Factories’ for the Production of Polyunsaturated Fatty Acids, Biotechnol. Adv. 18, 481–497.

    Article  Google Scholar 

  17. Sayanova, O., Smith, M.A., Lapinskas, P., Stobart, A.K., Dobson, G., Christie, W.W., Shewry, P.R., and Napier, J.A. (1997) Expression of a Borage Desaturase cDNA Containing an N-Terminal Cytochrome b5 Domain Results in the Accumulation of High Levels of Δ6-Desaturated Fatty Acids in Transgenic Tobacco, Proc. Nat. Acad. Sci. USA 94, 4211–4216.

    Article  PubMed  CAS  Google Scholar 

  18. Guil-Guerrero, J.L., Gómez-Mercado, F., García-Maroto, F., and Campra-Madrid, P. (2000) Occurrence and Characterization of Oils Rich in γ-Linolenic Acid Part I: Echium Seeds from Macaronesia, Phytochemistry 53, 451–456.

    Article  PubMed  CAS  Google Scholar 

  19. Guil-Guerrero, J.L., Gómez-Mercado, F., Rodríguez-García, I., Campra-Madrid, P., and García-Maroto, F. (2001) Occurrence and Characterization of Oils Rich in γ-Linolenic Acid (III): The Taxonomical Value of the Fatty Acids in Echium (Boraginaceae). Phytochemistry 58, 117–120.

    Article  PubMed  CAS  Google Scholar 

  20. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J., and Schilperoot, R.A. (1983) A Binary Plant Vector Strategy Based on Separation of Vir-and T-Region of the Agrobacterium tumefaciens Ti-Plasmid, Nature 303, 179–180.

    Article  CAS  Google Scholar 

  21. Schultz, L.D., Hofmann, K.J., Mylin, L.M., Montgomery, D.L., Ellis, R.W., and Hopper, J.E. (1987) Regulated Overproduction of the GAL4 Gene Product Greatly Increases Expression from Galactose-Inducible Promoters on Multi-Copy Expression Vectors in Yeast, Gene 61, 123–133.

    Article  PubMed  CAS  Google Scholar 

  22. Mylin, L.M., Hofmann, K.J., Schultz, L.D., and Hopper, J.E. (1990) Regulated GAL4 Expression Cassette Providing Controllable and High-Level Output from High-Copy Galactose Promoter in Yeast, Meth. Enzymol. 185, 297–309.

    Article  PubMed  CAS  Google Scholar 

  23. Ochman, H., Medhora, M., Garza, D., and Hartl, D.L. (1990) Amplification of Flanking Sequences by Inverse PCR, in PCR Protocols: A Guide to Methods and Applications (Innis, M.A., Gelfan, D.H., Sninsky, J.J., and White, T.J., eds.), pp. 219–227, Academic Press, London.

    Google Scholar 

  24. Thompson, J.D., Higgins, D.G., and Gibson, T.C. (1994) CLUSTALW: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, Nucl. Acids Res. 22, 4673–4680.

    PubMed  CAS  Google Scholar 

  25. Saitou, N., and Nei, M. (1987) The Neighbor-Joining Method: A New Method for Reconstructing Phylogenetic Trees, Mol. Biol. Evol. 4, 406–425.

    PubMed  CAS  Google Scholar 

  26. Page, R.D.M. (1996) TREEVIEW: An Application to Display Phylogenetic Trees on Personal Computers, Comput. Appl. Biosci. 12, 357–358.

    PubMed  CAS  Google Scholar 

  27. Taylor, B., and Powel, A. (1982) Isolation of Plant DNA and RNA, Focus 4, 4–6.

    Google Scholar 

  28. Chang, S., Puryear, J., and Cairney, J. (1993) A Simple and Efficient Method for Isolating RNA from Pine Trees, Plant Mol. Biol. Rep. 11, 113–116.

    CAS  Google Scholar 

  29. Shih, M.C., Lazan, G., and Goodman, H.M. (1986) Evidence in Favor of the Symbiotic Origin of Chloroplasts: Primary Structure and Evolution of Tobacco Glyceraldehyde-3-phosphate Dehydrogenases, Cell 47, 73–80.

    Article  PubMed  CAS  Google Scholar 

  30. Guerineau, F. (1995) Tools for Expressing Foreign Genes in Plants, Methods Mol. Biol. 49, 1–32.

    PubMed  CAS  Google Scholar 

  31. Bevan, M. (1984) Binary Agrobacterium Vectors for Plant Transformation, Nucl. Acids Res. 12, 8711–8721.

    PubMed  CAS  Google Scholar 

  32. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Wallroth, M., Eichholtz, D., Rogers, S.G., and Fraley, R.T. (1985) A Simple and General Method for Transferring Genes into Plants, Science 227, 1229–1231.

    Article  CAS  Google Scholar 

  33. Ito, H., Fukuda, K., Murata, K., and Kimura, A. (1983) Transformation of Intact Yeast Cells Treated with Alkali Cations, J. Bacteriol. 153, 163–168.

    PubMed  CAS  Google Scholar 

  34. Cesareni, G., and Murray, A.H. (1987) Plasmid Vectors Carrying the Replication Origin of Filamentous Single-Stranded Phages, in Genetic Engineering (Setlow, J.K., ed.), Vol. 9, pp. 135–154, Plenum Press, New York.

    Google Scholar 

  35. Rodríguez-Ruiz, J., Belarbi, E.-H., García Sánchez, J.L., and López Alonso, D. (1998) Rapid Simultaneous Lipid Extraction and Transesterification for Fatty Acid Analyses, Biotechnol. Tech. 12, 689–691.

    Article  Google Scholar 

  36. Napier, J.A., Sayanova, O., Sperling, P., and Heinz, E. (1999) A Growing Family of Cytochrome b5-Domain Fusion Proteins, Trends Plant Sci. 4, 2–4.

    Article  Google Scholar 

  37. Sperling, P., Zähringer, U., and Heinz, E. (1998) A Sphingolipid Desaturase from Higher Plants. Identification of a New Cytochrome b5 Fusion Protein, J. Biol. Chem. 273, 28590–28596.

    Article  PubMed  CAS  Google Scholar 

  38. Cahoon, F.B., Lindqvist, Y., Schneider, G., and Shanklin, J. (1997) Redesign of Soluble Fatty Acid Desaturases from Plants for Altered Substrate Specificity and Double Bond Position, Proc. Natl. Acad. Sci. USA 94, 4871–4877.

    Article  Google Scholar 

  39. Sayanova, O., Davies, G.M., Smith, M.A., Griffith, G., Stobart, A.K., Shewry, P.R., and Napier, J.A. (1999) Accumulation of Δ6-Unsaturated Fatty Acids in Transgenic Tobacco Plants Expressing a Δ6-Desaturated from Borago officinalis, J. Exp. Bot. 50, 1647–1652.

    Article  CAS  Google Scholar 

  40. Stymne, S., and Stobart, A.K. (1986) Biosynthesis of γ-Linolenic Acid in Cotyledons and Microsomal Preparations of the Developing Seeds of Common Borage (Borago officinalis), Biochem. J. 240, 385–393.

    PubMed  CAS  Google Scholar 

  41. Sayanova, O., Beaudoin, F., Libisch, B., Shewry, P., and Napier, J. (2000) Mutagenesis of the Borage Δ6 Fatty Acid Desaturase, Biochem. Soc. Trans. 28, 636–638.

    Article  PubMed  CAS  Google Scholar 

  42. Girke, T., Schmidt, H., Zähringer, U., Reski, R., and Heinz, E. (1998) Identification of a Novel Δ6-Acyl-Group Desaturase by Targeted Gene Disruption in Physcomitrella patens, Plant J. 15, 39–48.

    Article  PubMed  CAS  Google Scholar 

  43. 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.

    PubMed  CAS  Google Scholar 

  44. Cho, H.P., Nakamura, M.T., and Clarke, S.D. (1999) Cloning, Expression, and Nutritional Regulation of the Mammalian Δ-6 Desaturase, J. Biol. Chem. 274, 471–477.

    Article  PubMed  CAS  Google Scholar 

  45. Brosius, J. (1999) RNAs from All Categories Generate Retrosequences That May be Exapted as Novel Genes or Regulatory Elements, Gene 238, 115–134.

    Article  PubMed  CAS  Google Scholar 

  46. Sayanova, O., Napier, J., and Shewry, P.R. (1999) Δ6-Unsaturated Fatty Acids in Species and Tissues of the Primulaceae, Phytochemistry 52, 419–422.

    Article  CAS  Google Scholar 

  47. Guil-Guerrero, J.L., García-Maroto, F., Campra-Madrid, P., and Gómez-Mercado, F. (2000) Occurrence and Characterization of Oils Rich in γ-Linolenic Acid Part II: Fatty Acids and Squalene from Macaronesian Echium Leaves, Phytochemistry 54, 525–529.

    Article  PubMed  CAS  Google Scholar 

  48. Hofmann, K., and Stoffel, W. (1993) TMbase—A Database of Membrane Spanning Proteins Segments, Biol. Chem. Hoppe Seyler 374, 166.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departamento de Bioquímica, Facultad de Ciencias Experimentales, Universidad de Almería, E-04120, Almería, Spain

    Federico García-Maroto & José A. Garrido-Cárdénas

  2. Departamento de Biología Aplicada, Facultad de Ciencias Experimentales, Universidad de Almería, E-04120, Almería, Spain

    Juan Rodríguez-Ruiz, Miguel Vilches-Ferrón & Diego López Alonso

  3. Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, E-46100, Burjassot, Valencia, Spain

    Ana C. Adam & Julio Polaina

Authors
  1. Federico García-Maroto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. José A. Garrido-Cárdénas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Juan Rodríguez-Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Miguel Vilches-Ferrón
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ana C. Adam
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julio Polaina
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Diego López Alonso
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Diego López Alonso.

About this article

Cite this article

García-Maroto, F., Garrido-Cárdénas, J.A., Rodríguez-Ruiz, J. et al. Cloning and molecular characterization of the Δ6-desaturase from two Echium plant species: Production of GLA by heterologous expression in yeast and tobacco. Lipids 37, 417–426 (2002). https://doi.org/10.1007/s1145-002-0910-6

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s1145-002-0910-6

Keywords

Search

Navigation