These authors contributed equally to this work.
Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content
Article first published online: 8 JUL 2008
© 2008 Canadian Crown copyright. Journal compilation © 2008 Blackwell Publishing Ltd
Plant Biotechnology Journal
Volume 6, Issue 8, pages 799–818, October 2008
How to Cite
Xu, J., Francis, T., Mietkiewska, E., Giblin, E. M., Barton, D. L., Zhang, Y., Zhang, M. and Taylor, D. C. (2008), Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content. Plant Biotechnology Journal, 6: 799–818. doi: 10.1111/j.1467-7652.2008.00358.x
- Issue published online: 1 SEP 2008
- Article first published online: 8 JUL 2008
- Received 20 February 2008; accepted 21 May 2008.
- diacylglycerol acyltransferase 1 (DGAT1) gene;
- DGAT1 seed expression;
- mutant complementation;
- seed oil content;
- site-directed mutagenesis;
- Tropaeolum majus
A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 18.104.22.168) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATα quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of 14C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize 14C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%–10% on a dry weight basis, or a net increase of 11%–30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%–80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%–50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.