Cloning and molecular characterization of the acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) gene from Echium.
Boraginaceae species, such as those from the genus Echium, contain high levels of the Delta(6)-desaturated gamma-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 alpha-linolenic acid (18:3n-3) and gamma-linolenic acid over the dienoic linoleic acid (18:2n-6). Site-directed mutagenesis has revealed the presence of a critical residue (P(178) 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.
Mañas-Fernández
A
A
Laboratorio Genética III, Edificio CITE-II B, Universidad de Almería, 04120, Almería, Spain. fgmaroto@ual.es
Vilches-Ferrón
M
M
Garrido-Cárdenas
J A
JA
Belarbi
E-H
EH
Alonso
D L
DL
García-Maroto
F
F
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D051048:Diacylglycerol O-Acyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D036661:Echium / Q000201:enzymology* / Q000235:genetics*; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051048:Diacylglycerol O-Acyltransferase