Cloning And Primary Characterization Genes Of Diacylglycerol Acyltransferase(Dgat) In Soybean (Glycine Max)

Triacylglycerols (TAG) are the main storage lipids in most plants. Diacylglycerol acyl-transferase (DGAT) is the rate-limiting enzyme that catalyzes the final step in triacylglycerol (TAG) biosynthesis by converting diacylgycerol (DAG) and fatty acyl-coenzyme A (CoA) into triacylglycero1. Four different types of DGAT enzymes have been identified in various species, DGAT1, DGAT2, WS/DGAT and the soluble DGAT. DGAT1, DGAT2and WS/DGAT are all membrance binding proteins. DGAT proteins play an important role in TAG synthesize, seed development and maturing, plant senescence and stress response.In this paper, we used homology comparision with AtDGAT1, AtDGAT2protein amino acid sequence, finding two DGAT1, three DGAT2in soybean that share highly similarity with AtDGAT1, AtDGAT2, respectively. We cloned these five genes which we named as GmDGATl-1and GmDGAT1-2, three DGAT2were named as GmDGAT2-1, GmDGAT2-2and GmDGAT2-3. Analysis of the sequence reveals that these GmDGATs encode proteins of498,504,337,350and317animo acids, respectively. Phylogenetic analyses reveals that GmDGAT1-1and GmDGAT1-2proteins are members of the DGTA1protein family, shows a high similarity to DGAT1of higher plants like caster bean and tung tree. GmDGAT2-1, GmDGAT2-2, GmDGAT2-3belong to DGAT2protein family, have a high similarity with AtDGAT2, RcDGAT2and VfDGAT2. The analysis result of TMHMM2.0indicates that GmDGAT1-1and GmDGAT1-2proteins contain nine membrane spanning, GmDGAT2-1, GmDGAT2-2have two membrane spanning, while GmDGAT2-3presents three trans membrance domains.In order to study the subcelluar localization of GmDGAT, onion epidermal cells were cotransformed transiently with mCherry-HDEL and GmDGATs-GFP, the fluorescence suggested that GmDGATs localized in endoplasmic reticulum and nucleolus.To investigate the functional properties of GmDGAT enzymes, yeast mutant strain H1246(dgal, lrol, arel, are2) quadruple mutants were used to detected they DGAT activities, the result shows that GmDGAT1-1,GmDGAT1-2,GmDGAT2-land GmDGAT2-2all have activities.To investigate how the expression of GmDGATs is regulated in different organs of soybean and the expression patterns of GmDGATs under salt stress, Real-Time PCR were used to detected these response. Results showed that the expression level of GmDGAT1-1was highest in immature seed, and that of GmDGATl-2has a relatively high transcript levels in flowers, stem, roots and leaves. GmDGAT2-1has a low expression levels in seeds, comparable to stem, roots, and leaves, a higher level in flowers. GmDGAT2-2present a totally opposite expression mode compared to GmDGAT2-1, has a low expression levels in stem, leaves and flowers, a higher level in matured seeds.While GmDGAT2-3has an old leave-specific expression pattern. The expression level of GmDGAT1-1, GmDGAT2-2and GmDGAT2-3were up regulated after a250mM NaCl salt treatment. Compared with those three above-mentioned genes, GmDGAT1-2and GmDGAT2-1had a slight fluctuation during the salt treatment, and then come back to initial expression level after6hours.To investigate the functions of the GmDGAT, we identified Arabidopsis thaliana mutant dgatl, dgat2, five over expression vector Super-1300-GmDGATs were constructed to introducing Arabidopsis. Up to now, three lines of transgenic Super-1300, two lines of transgenic Super-1300-GmDGAT1-1, four lines of transgenic Super-1300-GmDGAT2-1, two lines and six lines of transgenic Super-1300-GmDGAT2-2, Super-1300-GmDGAT2-3have obtained respectively, all of these work provided the foundation for further investigation on GmDGATs.