Cloning and functional characterization of a Type 3 Diacylglycerol Acyltransferasegene (GmDGAT3-2) from soybean (Glycine max L.)
Creators: name: Xue, Jinai
name: Gao, Huiling
name: Xue, Yinghong
name: Shi, Ruixiang
name: Liu, Mengmeng
name: Han, Lijun
name: Gao, Yu
name: Zhou, Yali
name: Zhang, Fei
name: Zhang, Haiping
name: Li, Runzhi
Publisher: name: Zenodo
Year: 2022

description: Diacylglycerol acyltransferases (DGAT) function as the key rate-limiting enzymes in <em>de novo</em> biosynthesis of triacylglycerol (TAG) by transferring an acyl group from acyl-CoA to <em>sn</em>-3 of diacylglycerol (DAG) to form TAG. Here, two members of <em>type 3</em> <em>DGAT</em><em> </em>gene family, <em>GmDGAT3-1</em> and <em>GmDGAT3-2</em>, were identified from soybean (<em>Glycine max</em>) 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 <em>GmDGAT3-1</em>'s in various soybean tissues such as leaves, flowers and seeds. Functional complementation assay using TAG-deficient yeast (<em>Saccharomyces cerevisiae</em>) mutant H1246 demonstrated that <em>GmDGAT3-2</em> 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 (<em>Nicotiana benthamiana</em>) data showed that heterogeneous expression of <em>GmDGAT3-2</em> 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, <em>GmDGAT3-2</em> 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.
description: These two <em><strong>GmDGAT3 </strong></em>genes were identified from the soybean genome in Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html#!info? Alias =Org _Gmax). The accession number of <em>GmDGAT3-1</em> is Glyma.13G118300.1 (https://phytozome-next.jgi.doe.gov/report/gene/Gmax_ Wm82_a2_v1/Glyma.13G118300). The accession number of <em>GmDGAT3-2</em> is Glyma.17G041600.1 (https://phytozome-next.jgi.doe.gov/report/gene/Gmax_Wm82_a2_v1/Glyma.17G041600 ) A phylogenetic tree of GmDGAT3 and other DGAT3 proteins was constructed using the neighbor-joining method in the MEGA 6.0 (see Table S1 for protein information derived from different species and the sequence downloaded from NCBI (https://www.ncbi.nlm.nih.gov) ). All experiments were carried out at least in triplicate. The data was analyzed using IBS SPSS Statistics software Version 22. Duncan's test was employed to detect the statistical significance (P &lt; 0.05) when comparing differences between two groups.