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Cloning and functional characterization of a Type 3 Diacylglycerol Acyltransferasegene (GmDGAT3-2) from soybean (Glycine max L.)

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Apr 29, 2022 version files 12.62 KB

Abstract

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.