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Diacylglycerol acyltransferases (DGAT) function as the key rate-limiting
enzymes in de novo biosynthesis of triacylglycerol (TAG) by transferring
an acyl group from acyl-CoA to sn-3 of diacylglycerol (DAG) to form TAG.
Here, two members of type 3 DGAT gene family, GmDGAT3-1 and GmDGAT3-2,
were identified from soybean (Glycine max) genome. Both of them were
predicted to encode soluble cytosolic proteins containing the typical
thioredoxin-like ferredoxin domain. Quantitative PCR analysis revealed
that GmDGAT3-2 expression was much higher than GmDGAT3-1’s in various
soybean tissues such as leaves, flowers and seeds. Functional
complementation assay using TAG-deficient yeast (Saccharomyces cerevisiae)
mutant H1246 demonstrated that GmDGAT3-2 fully restored TAG biosynthesis
in the yeast and preferentially incorporated monounsaturated fatty acids
(MUFAs), especially oleic acid (C18:1) into TAGs. This substrate
specificity was further verified by feeding assays and in vitro enzyme
activity characterization. Notably, transgenic tobacco (Nicotiana
benthamiana) data showed that heterogeneous expression of GmDGAT3-2
resulted in significant increase of seed oil and C18:1 levels, but little
change in contents of protein and starch compared to the EV-transformed
tobacco plants. Taken together, GmDGAT3-2 displayed a strong enzymatic
activity to catalyze TAG assembly with high substrate specificity for
MUFAs, particularly C18:1, playing an important role in the cytosolic
pathway of TAG synthesis in soybean. The present findings provide a
scientific reference for improving oil yield and FA composition in soybean
through gene modification, further expanding our knowledge of TAG
biosynthesis and its regulatory mechanism in oilseeds.
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