Skip to main content
Log in

Transgenic barley producing essential polyunsaturated fatty acids

  • Original Papers
  • Published:
Biologia Plantarum

Abstract

Polyunsaturated fatty acids (PUFAs) affect diverse physiological processes and human health. Most cereals are poor in n-3 and n-6 PUFAs. Using biolistics, barley (Hordeum vulgare L. cv. Golden Promise) was transformed with an artificial gene encoding Δ6-desaturase (D6D) under an endosperm-specific promoter. This artificial gene was designed from the sequence of D6D of the filamentous fungus Thamnidium elegans, but codon usage was optimised for cereals. A signal sequence from the gene encoding for high molecular mass glutenin Dx5 was added to a destinate mature protein. Successful transformation was confirmed in T0 plants at the genomic level and in T1 seeds at the transcriptomic and metabolomic levels. Transformed plants produced up to 0.141 % of γ-linolenic acid (GLA) and 0.294 % of stearidonic acid (SDA) of the total amount of fatty acids in their grains. Although the content of these fatty acids was relatively low, the current study provides the first evidence that transgenic barley can be a source of GLA/SDA.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

Abbreviations

ALA:

α-linolenic acid

D6D:

Δ6-desaturase

DGLA:

dihomo-γ-linoleic acid

HMM:

high-molecular-mass

GAPDH:

glyceraldehyde 3-phosphate dehydrogenase

GLA:

γ-linolenic acid

LA:

linoleic acid

OA:

oleic acid

PUFA:

polyunsaturated fatty acid

SDA:

stearidonic acid

References

  • Anderson, O.D., Greene, F.C., Anderson, O.D., Greene, F.C., Yip, R.E., Halford, N.G., Shewry, P.R., Malpica-Romero, J.M.: Nucleotide sequences of the two high-molecularweight glutenin genes from the D-genome of a hexaploid bread wheat, Triticum aestivum L. cv Cheyenne. — Nucl. Acids Res. 17: 461–462, 1989.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Brenner, R.R.: Regulatory function of D6-desaturase — a key enzyme of polyunsaturated fatty acid synthesis. — Adv. exp. Med. Biol. 83: 85–101, 1976.

    Article  Google Scholar 

  • Certík, M., Adamechova, Z.: Cereal-based bioproducts containing polyunsaturated fatty acids. — Lipid Technol. 21: 250–253, 2009.

    Article  CAS  Google Scholar 

  • Certik, M., Adamechova, Z., Slavikova, L.: Biotechnological enrichment of cereals with polyunsaturated fatty acids. — In: Hou, C.T., Shaw, J.F. (ed.): Biocatalysis and Biomolecular Engineering. Pp. 175–193. John Wiley & Sons, Hoboken 2010.

    Chapter  Google Scholar 

  • Certik, M., Sakuradani, E., Shimizu, S.: Desaturase-defective fungal mutants: useful tools for the regulation and overproduction of polyunsaturated fatty acids. — Trends Biotechnol. 16: 500–505, 1998.

    Article  CAS  Google Scholar 

  • Certik, M., Shimizu, S.: Biosynthesis and regulation of microbial polyunsaturated fatty acid production. — J. Biosci. Bioeng. 87: 1–14, 1999.

    Article  PubMed  CAS  Google Scholar 

  • Cheawchanlertfa, P., Cheevadhanarak, S., Tanticharoen, M., Maresca, B., Laoteng, K.: Up-regulated expression of desaturase genes of Mucor rouxii in response to low temperature associates with pre-existing cellular fatty acid constituents. — Mol. Biol. Rep. 38: 3455–3462, 2011.

    Article  PubMed  CAS  Google Scholar 

  • Chen, R., Tsuda, S., Matsui, K., Mizutani, M.F., Ochiai, M., Shimizu, S., Sakuradani, E., Aoki, T., Imaizumi, R., Ayabe, S., Tanaka, Y.: Production of γ-linolenic acid in Lotus japonicus and Vigna angularis by expression of the Δ-6-fatty-acid desaturase gene isolated from Mortirella alpina. — Plant Sci. 169: 599–605, 2005.

    Article  CAS  Google Scholar 

  • Christensen, A.H., Quail, P.H.: Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. — Transgenic Res. 5: 213–218, 1996.

    Article  PubMed  CAS  Google Scholar 

  • Dahleen, L.S., Manoharan, M.: Recent advances in barley transformation. — In Vitro cell. dev. Biol. Plant 43: 493–506, 2007.

    Article  CAS  Google Scholar 

  • Flider, F.J.: GLA: uses and new sources. — Inform 16: 279–282, 2005.

    Google Scholar 

  • Furtado, A., Henry, R.J., Pellegrineschi, A.: Analysis of promoters in transgenic barley and wheat. — Plant Biotechnol. J. 7: 240–253, 2009.

    Article  PubMed  CAS  Google Scholar 

  • García-Maroto, F., Garrido-Cárdenas, J.A., Rodríguez-Ruiz, J., Vilches-Ferrón, M., Adam, A.C., Polaina, J., López Alonso, D.: 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.

    Article  PubMed  Google Scholar 

  • Gill, I., Valivety, R.: Polyunsaturated fatty acids, part 1: occurrence, biological activities and applications. — Trends Biotechnol. 15: 401–409, 1997.

    Article  PubMed  CAS  Google Scholar 

  • Gregova, E., Mihalik, D., Slikova, S., Sramkova, Z.: Allelic variation of HMW glutenin subunits and 1BL. 1RS translocation in Slovak common wheats. — Cereal Res. Commun. 35: 1675–1683, 2007.

    Article  CAS  Google Scholar 

  • Gubisova, M., Mihalik, D., Konopkova, L.: Regeneration efficiency of Slovak spring barley cultivars and winter wheat cultivars. — Agriculture 57: 76–83, 2011.

    Google Scholar 

  • Gunstone, F.D.: Gamma linolenic acid — occurrence and physical and chemical properties. — Progr. Lipid Res. 31: 145–161, 1992.

    Article  CAS  Google Scholar 

  • Harwood, W.A., Smedley, M.A.: Barley transformation using biolistic techniques. — In: Jones, H.D., Shewry, P.R. (ed.): Methods in Molecular Biology. Transgenic Wheat, Barley and Oats. Pp. 125–136. Humana Press, New York 2009.

    Chapter  Google Scholar 

  • He, G.Y., Jones, H.D., D’Ovidio, R., Masci, S., Chen, M., West, J., Butow, B., Anderson, O.D., Lazzeri, P., Fido, R., Shewry, P.R.: Expression of an extended HMW subunit in transgenic wheat and the effect on dough mixing properties. — J. Cereal Sci. 42: 225–231, 2005.

    Article  CAS  Google Scholar 

  • Hong, H., Datla, N., Reed, D.W., Covello, P.S., MacKenzie S.L., Qiu, X.: High-level production of γ-linolenic acid in Brassica juncea using a D6 desaturase from Pythium irregulare. — Plant Physiol. 129: 354–362, 2002.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Hudson, B.J.: Evening primrose (Oenothera spp.) oil and seed. — J. amer. Oil Chem. Soc. 61: 540–542, 1984.

    Article  CAS  Google Scholar 

  • Liu, J.W., DeMichele, S., Bergana, M., Bobik, E., Hastilow, C., Chuang, L.T., Mukerji, P, Huang, Y.S.: Characterization of oil exhibiting high gamma-linolenic acid from a genetically transformed canola strain. — J. amer. Oil Chem. Soc. 78: 489–493, 2001.

    Article  CAS  Google Scholar 

  • Matsumoto, T., Tanaka, T., Sakai, H., Amano, N., Kanamori, H., Kurita, K., Kikuta, A., Kamiya, K., Yamamoto, M., Ikawa, H., Fujii, N., Hori, K., Itoh, T., Sato, K.: Comprehensive sequence analysis of 24,783 barley fulllength cDNAs derived from 12 clone libraries. — Plant Physiol. 156: 20–28, 2011.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Na-Ranong, S., Laoteng, K., Kittakoop, P., Tanticharoen, M., Cheevadhanarak, S.: Substrate specificity and preference of delta-6-desaturase of Mucor rouxii. — FEBS Lett. 579: 2744–2748, 2005.

    Article  PubMed  CAS  Google Scholar 

  • Nykiforuk, C.L., Shewmaker, C., Harry, I., Yurchenko, O.P., Zhang, M., Reed, C., Oinam, G.S., Zaplachinski, S., Fidantsef, A., Boothe, J.G., Moloney, M.M.: High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds. — Transgenic Res. 21: 367–381, 2012.

    Article  PubMed  CAS  Google Scholar 

  • Puigbó, P., Guzmán, E., Romeu, A., Garcia-Vallvé, S.: OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. — Nucl. Acids Res. 35: 126–131, 2007.

    Article  Google Scholar 

  • Ratledge, C.: Fatty acid biosynthesis in microorganisms being used for single cell oil production. — Biochimie 86: 807–815, 2004.

    Article  PubMed  CAS  Google Scholar 

  • Reddy, A.S., Nuccio, M.L., Gross, L.M.: Isolation of a D6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC6803 by gain-of-function expression in Anabaena sp. strain PCC7120. — Plant mol. Biol. 22: 293–300, 1993.

    Article  PubMed  CAS  Google Scholar 

  • Reddy, A.S., Thomas, T.L.: Expression of a cyanobacterial D6-desaturase gene results in Γ-linolenic acid production in transgenic plants. — Nat. Biotech. 14: 639–642, 1996.

    Article  CAS  Google Scholar 

  • Sato, S., Xing, A., Ye, X., Schweiger, B., Kinney, A., Graef, G., Clemente, T.: Production of Γ-linolenic acid and stearidoinic acid in seeds of marker-free transgenic soybean. — Crop Sci. 44: 646–652, 2004.

    Article  CAS  Google Scholar 

  • Sayanova, O., Smith, M.A., Lapinskas, P., Stobart, A.K., Dobson, G., Christie, W.W., Shewry, P.R., Napier, J.A.: Expression of borage desaturase cDNA containing an Nterminal cytochrom b5 domain results in the accumulation of high levels of D6-desaturated fatty acids in transgenic tobacco. — Proc. nat. Acad. Sci. USA 94: 4211–4216, 1997.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Sood, P., Bhattacharya, A., Sood, A.: Problems and possibilities of monocot transformation. — Biol. Plant. 55: 1–15, 2011.

    Article  CAS  Google Scholar 

  • Stymne, S., Stobart, A. K.: Biosynthesis of γ-linolenic acid in cotyledons and microsomal preparation of the developing seeds of common borage (Borago officinalis). — Biochem. J. 24: 385–393, 1986.

    Google Scholar 

  • Tang, G.Y., Wei, L.Q, Liu, Z.J., Bi, Y.P., Shan, L.: Ectopic expression of peanut acyl carrier protein in tobacco alters fatty acid composition in the leaf and resistance to cold stress. — Biol. Plant. 56: 493–501, 2012.

    Article  CAS  Google Scholar 

  • Traitler, H., Wille, H.J., Studer, A.: Fractionation of black currant seed oil. — J. amer. Oil Chem. Soc. 65: 755–760, 1988.

    Article  CAS  Google Scholar 

  • Ucciani, E.: Potential sources of gamma-linolenic acid. — OCL. Oleagineux Corps Gras Lipides 2: 319–322, 1995.

    CAS  Google Scholar 

  • Wang, D., Li, M., Wei, D., Cai, Y., Zhang, Y., Xing, L.: Identification and functional characterization of the delta 6-fatty acid desaturase gene from Thamnidium elegans. — J. Eukaryot. Microbiol. 54: 110–117, 2007.

    Article  PubMed  CAS  Google Scholar 

  • Zhao, X., Kong, X., Hua, Y., Feng, B., Zhao, Z.K.: Medium optimization for lipid production through co-fermentation of glucose and xylose by oleaginous yeast Lipomyces starkeyi. — Eur. J. Lipid Sci. Technol. 110: 405–412, 2008.

    Article  CAS  Google Scholar 

  • Zhou, X., R., Robert, S., Singh, S., Green, A.: Heterelogous production of GLA and SDA by expression of an Echium plantagineum Δ-6 desaturase gene. — Plant Sci. 170: 665–673, 2006.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to D. Mihálik.

Additional information

Acknowledgements: This work was supported by the Slovak Research and Development Agency (the contract Nos. APVV-0294-11 and APVV-0662-11). We would like to thank Dr. H.D. Jones for providing the pLRPT vector.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mihálik, D., Gubišová, M., Klempová, T. et al. Transgenic barley producing essential polyunsaturated fatty acids. Biol Plant 58, 348–354 (2014). https://doi.org/10.1007/s10535-014-0406-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10535-014-0406-9

Additional key words

Navigation