Cloning And Functional Characterization Of GmPDAT1-B And GmDGAT3-2 Genes In Glycine Max

Soybean(Glycine max L.Merr),one of the major grain and oil crops in the world,produces 40%protein and 20% oil in its seeds,respectively.Soybean oil consumption accounts for more than one-third of the global vegetable oil market.With the increasing demand for soybean oil as edible oils,biofuels and other oleochemicals,developing high-oil varieties and enhancing soybean oil yield are the main fields for soybean genetic breeding and production.In-depth analysis of the regulation mechanism of soybean fatty acid/oil biosynthesis and functional characterization of key genes involved in lipid synthesis can provide scientific basis for genetic improvement of soybean oil yield and quality as well as molecular targets for oil metabolic engineering..The most majority of oils stored in plant seeds are triacylglycerols(TAG).The key rate-limiting enzymes for the final acylation reaction in the TAG synthesis are mainly two enzyme systems,namely acyl-Co A-dependent diacylglycerol acyltransferases(DGAT,including DGAT1,DGAT2 and DGAT3)and acyl-Co A-independent phospholipids:diacylglycerol acyltransferase(PDAT,including PDAT1 and PDAT2).DGAT transfers acyl group bound to Co A molecule into the sn-3 position of the sn-1,2-diacylglycerol(DAG)to generate TAG.However,PDAT removes acyl group from phosphatidylcholine(PC)molecule to the sn-3 position of DAG to form TAG.Members of the soybean DGAT1 and DGAT2 families were functionally identified in previous reports.However,little information is available for characterization of soybean DGAT3 and PDAT1 family members and their functions..Therefore,this study was conducted for genome-wide identification of soybean GmDGAT3 and GmPDAT1 family members,followed by quantitative analysis of their tissue-specific expression profiles.The ORF sequences of GmDGAT3-2 and GmPDAT1-B genes highly expressed in developing soybean seeds were cloned,respectively,and thentheir constitutive expression vectors in plant and yeast were separately constructed.Yeast functional analysis and tobacco transient expression were performed to investigate GmDGAT3-2 and GmPDAT1-B enzymatic activities and their biological functions by examining their impacts on TAG synthesis and fatty acid compositions in the host cells a Finally,a number of new knowledge were obtained,providing insight into further understanding of molecular mechanism underlying soybean oil biosynthesis and regulation,and also benefiting the development of optimized strategy for oil metabolic engineering in soynean and other oil crops.The main findings are as follows:1.BLAST analysis of the soybean genome database was performed by Arabidopsis thaliana At PDAT1 and peanut Ah DGAT3 sequences as probes,respectively.Six GmPDAT1 genes(named GmPDAT1-A~F)and two GmDGAT3 genes(GmDGAT3-1 and GmDGAT3-2)in the soybean genome were identified.Multiple sequence alignment analysis showed that both GmPDAT1 s and GmDGAT3 s had the substrate-binding site of acyltransferase,with GmPDAT1 s containing five typical conserved domains of PDAT enzyme family,and GmDGAT3 s having six typical conserved domains of DGAT3 enzyme family.Phylogenetic analysis indicated that GmPDAT1 s were classified into three subgroups,with highly similarity to PDAT enzyme protein from different species.Whereas,two GmDGAT3 enzyme proteins were grouped together,showing highly similarity to the DGAT3 enzyme proteins of Mucuna pruriens and Cajanus cajan.2.The gene expression profiles of six GmPDAT1 s and two GmDGAT3 s in different tissues and organs of soybean(cv.Jack)were examined by RT-PCR and real-time PCR(q RT-PCR).GmPDAT1 s and GmDGAT3 s were detected to expressin roots,stems,flowers,leaves,pods and developing seeds of soybean,but the expression levels were significantly different.GmPDAT1-B and GmDGAT3-2 are highly expressed in developing seeds.3.The coding sequences of GmPDAT1-B and GmDGAT3-2 were successfully cloned from soybean development seeds using high-fidelity RT-PCR.The yeast expression vector of GmPDAT1-B and GmDGAT3-2(p YES2-GmPDAT1-B/GmDGAT3-2)was constructed respectively,and then transformed into yeast(Saccharomyces cerevisiae)mutant H1246 cells(deficient in TAG synthesis).Analysis of total oil and fatty acid composition in the positive-transgenic yeast cell lines showed that overexpression of GmPDAT1-B or GmDGAT3-2 both restored the TAG synthesis in yeast mutant H1246 cells.Compared with the empty-vector transferred H1246 cells,the oil content in the transgenic yeast cells of GmPDAT1-B or GmDGAT3-2 were increased by 1.66 and 1.42 folds,respectively.Higher levels of saturated fatty acids(16:0,palmitic acid,and 18:0,stearic acid)were synthesized and accumulated in the transgenic yeast cells of GmPDAT1-B,whereas larger amount of monounsaturated fatty acids(16:1,palmitoleic acid,and 18:1,oleic acid)were produced in the GmDGAT3-2 transgenic yeast cells.This indicates that the two acyltransferases have different substrate preferences for various acyl groups.4.Plant constitutive expression vectors of GmPDAT1-B and GmDGAT3-2(p CAMBIA1303-GmPDAT1-B/GmDGAT3-2)were separately constructed.Agrobacterium-mediatedinfiltration was used to infect tobacco leaf for transient expression of GmPDAT1-B or GmDGAT3-2,respectively.The tobacco leaves infected with empty vector were used as control.RNA examination showed that GmPDAT1-B and GmDGAT3-2 genes were highly expressed in the transgenic tobacco leaves.The determination of oil content and fatty acid composition in the transient-expressed tobacco leaf tissues showed that GmPDAT1-B and GmDGAT3-2 could significantly promote the synthesis and accumulation of oil compared to the control.The total oil content was 4.15 and 3.24 times higher,respectively,in the transient-expressed tobacco leaves of GmPDAT1-B and GmDGAT3-2 than that of the uninfected and empty-vector controls(p<0.05).In the GmPDAT1-B transient-expressed leaves,palmitic acid(16:0)decreased by 8.20%,but stearic acid(18:0)increased by 7.62%,and oleic acid(18:1)also increased significantly(p<0.05).In the GmDGAT3-2 transient-expressed leaves,palmitic acid level reduced by11.67% whereas oleic acid content enhanced significantly by 12.34%(p<0.05).This demonstrates that GmPDAT1-B and GmDGAT3-2 have a higher substrate preference for 18:0 and 18:1,respectively.In summary,this study was the first to clone and identify soybean GmPDAT1-B and GmDGAT3-2genes encoding the enzyme with higher acyltransferase activity in catalyzing TAG synthesis.Moreover,GmDGAT3-2 is the second soluble enzyme protein identified to have DGAT activity after functional characterization of peanut Ah DGAT3.GmPDAT1-B and GmDGAT3-2 genes can be used as the molecular targets for soybean oil genetic improvement,which can also be used for transgenic breeding practice to improve the oil yield and quality in other oil plants.