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Cloning and Molecular Characterization of the Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) Gene from Echium

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Lipids

Abstract

Boraginaceae species, such as those from the genus Echium, contain high levels of the Δ6-desaturated γ-linolenic (18:3n-6) and octadecatetraenoic (18:4n-3) acids. These are unusual fatty acids among the plant kingdom that are gaining interest due to their benefits to human health. The potential utility of acyltransferases aimed at an increase in oil yield and fatty acid profiling has been reported. In this work, a gene encoding an acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) was cloned from Echium pitardii. Genomic and cDNA sequences obtained revealed a gene structure composed of 16 exons, yielding a protein (EpDGAT) of 473 amino acids with high similarity to DGAT1 enzymes of plants. Protein features such as a predicted structure with a highly hydrophilic N-terminus followed by 10 transmembrane domains, as well as the presence of diverse specific signatures, also indicate that EpDGAT belongs to the DGAT1 family. indeed. DGAT activity of the protein encoded by EpDGAT was confirmed by heterologous expression of the full-length cDNA in a yeast mutant (H1246) defective in the synthesis of triacylglycerols. Fatty acid composition of the triacylglycerols synthesized by EpDGAT in H1246 yeast cultures supplemented with polyunsaturated fatty acids suggest a substrate preference for the trienoic fatty acids α-linolenic acid (18:3n-3) and γ-linolenic acid over the dienoic linoleic acid (18:2n-6). Site-directed mutagenesis has revealed the presence of a critical residue (P178 in EpDGAT) within a reported thiolase signature for binding of acyl-enzyme intermediates that might be involved in the active site of the enzyme. Transcript analysis for EpDGAT shows an ubiquitous expression of the gene which is increased in leaves during senescence.

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Abbreviations

ALA:

Alpha-linolenic acid

cDNA:

Complementary DNA

CTAB:

Cetyl trimethylammonium bromide

DAG:

Diacylglycerol

DGAT:

Acyl-CoA:diacylglycerol acyltransferase

DIG:

Digoxigenin

FFA:

Free fatty acids

GC:

Gas chromatography

GLA:

Gamma-linolenic acid

IPCR:

Inverse PCR

LNA:

Linoleic acid

NL:

Neutral lipids

PCR:

Polymerase chain reaction

PL:

Polar lipids

PUFA:

Polyunsaturated fatty acid

RT-PCR:

Reverse transcriptase PCR

SDS:

Sodium dodecylsulfate

SE:

Steryl esters

ST:

Sterols

TAG:

Triacylglycerol

TL:

Total lipids

TLC:

Thin layer chromatography

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Acknowledgments

This work was supported by grants from the Ministerio de Ciencia y Tecnología (MCYT, AGL2005-01498/AGR) and Junta de Andalucía (P05-189-AGR). J.A. Garrido-Cárdenas and A. Mañas-Fernández were recipients of postgraduate fellowships from the MCYT and Junta de Andalucía, respectively. We are also grateful to J. Pérez-Parra and J.C. Gázquez for providing greenhouse facilities and technical assistance in plant culture at the “Estación Experimental Las Palmerillas (CAJAMAR)”. Dr. Stymne (Swedish University of Agricultural Sciences, Uppsala) kindly provided the mutant strain of S. cerevisiae H1246, and is gratefully acknowledged.

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Authors and Affiliations

  1. Laboratorio Genética III, Edificio CITE-II B, Universidad de Almería, 04120, Almería, Spain

    A. Mañas-Fernández, M. Vilches-Ferrón, J. A. Garrido-Cárdenas, D. L. Alonso & F. García-Maroto

  2. Departamento Ingeniería Química, Universidad de Almería, 04120, Almería, Spain

    E.-H. Belarbi

Authors
  1. A. Mañas-Fernández
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  2. M. Vilches-Ferrón
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  3. J. A. Garrido-Cárdenas
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  5. D. L. Alonso
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  6. F. García-Maroto
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Correspondence to F. García-Maroto.

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11745_2009_3303_MOESM1_ESM.doc

Deduced donor and acceptor splice sites of the EpDGAT gene. Splicing sites were deduced from the comparison between genomic and cDNA sequences. Non-standard acceptor sequence on intron 3 is underlined. (DOC 27 kb)

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Mañas-Fernández, A., Vilches-Ferrón, M., Garrido-Cárdenas, J.A. et al. Cloning and Molecular Characterization of the Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) Gene from Echium . Lipids 44, 555–568 (2009). https://doi.org/10.1007/s11745-009-3303-9

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