Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis.
Vernonia galamensis accumulates vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) as the major fatty acid in its seed oil. Such epoxy fatty acids are useful in a number of industrial applications. Successful genetic engineering of commercial oilseed crops to produce high levels of vernolic acid depends on a better understanding of the source plant enzymes for vernolic acid accumulation. Developing V. galamensis seed microsome assays demonstrate that diacylglycerol acyltransferase (DGAT), an enzyme for the final step of triacylglycerol synthesis, has a strong substrate preference for vernolic acid bearing substrates including acyl-CoA and diacylglycerol. There are two classes of DGATs known as DGAT1 and DGAT2. Here we report on the isolation, characterization, and functional analysis of two DGAT1 cDNAs from V. galamensis (VgDGAT1a and VgDGAT1b). VgDGAT1a and VgDGAT1b are expressed in all plant tissues examined with highest expression in developing seeds. Enzymatic assays using isolated microsomes from transformed yeast show that VgDGAT1a and VgDGAT1b have the same DGAT activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-dioleoylglycerol are preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. This data indicates that the two VgDGAT1s are functional, but not likely to be responsible for the selective accumulation of vernolic acid in V. galamensis seed oil.
Yu
Keshun
K
Department of Plant and Soil Science, University of Kentucky, 1405 Veterans Drive, Lexington, KY 40546-0091, USA.
Li
Runzhi
R
Hatanaka
Tomoko
T
Hildebrand
David
D
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D051048:Diacylglycerol O-Acyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004852:Epoxy Compounds / Q000737:chemistry; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009829:Oleic Acids / Q000737:chemistry; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012639:Seeds / Q000201:enzymology*; D016415:Sequence Alignment; D035741:Vernonia / Q000737:chemistry / Q000201:enzymology*
D018076:DNA, Complementary; D004852:Epoxy Compounds; D009829:Oleic Acids; D010940:Plant Proteins; C081136:vernolic acid; D051048:Diacylglycerol O-Acyltransferase