The Role Of DGAT And PDAT Genes Involved In α-linolenic Acid Accumulation Of Tree Peony Seeds

Tree peony is a perennial deciduous shrub in Paeoniaceae Section Mouton DC which originated from China.Tree peony seed oil is rich in polyunsaturated fatty acids,especiallyα-linolenic acid(ALA)is more than 40%,that is an important garden flower and medicinal plant with nutritional characteristics.Typically,the final step in TAG biosynthesis is catalyzed by diacylglycerol acyltransferases(DGAT)and phospholipid: diacylglycerol acyltransferases(PDAT).DGAT and PDAT can selectively transfer α-linolenic acid to glycerol,which directly affects the composition and accumulation of lipid.Based on the exsiting reports and research of our group,it is suggested that the sn-3 of tree peony lipid is probably the main binding site of α-linolenic acid,which indicates that DGAT and PDAT are probably involved in the efficient accumulation of α-linolenic acid in tree peony.In this study,five acyltransferase unigenes were sreened from the transcriptome database of tree peony(Paeonia rockii).We cloned these five genes and analysed their characteristics.Then the enzyme activity and substrate specificity of Pr DGATs and Pr PDATs were measured by the lipotoxicity rescue assay of TAG synthesis-deficient yeast.Moreover,the target genes were transiently expressed in tobacco(Nicotiana benthamiana)leaves and the TRV-VIGS were preformed to identify their functions in the accumulation of α-linolenic acid.The study aims to provide the basis for revealing the regulation mechanism of high-level accumulation of α-linolenic acid in tree peony seeds,and also provide valuable genes for improving fatty acid compositions and molecular breeding of tree peony.The main results were as follows:1.Five genes encoding diacylglycerol acyltransferases were cloned,named Pr DGAT1,Pr DGAT2,Pr PDAT1-1,Pr PDAT1-2,Pr PDAT2,and the lengths of their coding regions were 1554 bp,981bp,2019 bp,2028bp,2040 bp,respectively.Moreover,their open reading frame(ORF)encoded 517,326,672,675,679 amino acids,respectively.They all have a conserved domain sequence.The conserved domain of Pr DGAT1 and Pr DGAT2 belongs to the MBOAT superfamily and LPLAT superfamily,respectively.And the conserved domains of Pr PDAT1-1,Pr PDAT1-2,and Pr PDAT2 all belong to the phosphatidylcholine-sterol O-acyltransferase superfamily.Phylogenetic analysis suggested that Pr DGAT1-1 is highly homologous to DGAT1 of Olea europaea and camellia oleifera.Pr DGAT2 is highly homologous to DGAT2 of Quercus suber and Mucuna pruriens.Pr PDAT1-1 is highly homologous to PDAT1 of Vitis vinifera and Sesamum indicum.Pr PDAT1-2 is highly homologous to PDAT1 of Theobroma cacao and Rosa chinensis.Pr PDAT2 is highly homologous to PDAT of Actinidia chinensis and Prunus sibirica.By subcellular localization analysis,they were all localized on the endoplasmic reticulum.2.q RT-q PCR analysis showed that five genes encoding diacylglycerol acyltransferases were all expressed in the whole stages.However there were significant differences in expression levels at different stages.The expression patterns of Pr DGAT1,Pr DGAT2 and Pr PDAT1-2 are similar.The relative expression level of Pr PDAT1-1 is stable throughout the developmental stage of the seeds.Only Pr PDAT1-1 was expressed at a relatively high level in the early stages of seed development(S1-S3),but the relative expression levels of the five genes were higher in the later stage,which may indicate that the later stages of seed development(S4-S5)are the key periods for lipid synthesis and α-linolenic acid accumulation.2.The Pr DGAT1,Pr DGAT2,Pr PDAT1-1,Pr PDAT1-2,and Pr PDAT2 genes were expressed in quadruple yeast mutants with defective TAG biosynthesis.The lipotoxicity rescue assay showed that in the yeast expression system,except for Pr PDAT1-1,which had no enzyme activity or could not transfer C18: 1,C18: 2,C18: 3 fatty acids,all other enzymes had enzyme activity.Specifically,Pr DGAT1 can transfer C18: 1,C18: 2,C18: 3fatty acids;Pr DGAT2 can transfer C18: 2,C18: 3 fatty acids;Pr PDAT1-2 prefers to transfer C18: 1 fatty acids;Pr PDAT2 prefers to transfer C18: 3 fatty acids.3.Pr DGAT1,Pr DGAT2,Pr PDAT1-1,Pr PDAT1-2 and Pr PDAT2 were transiently expressed in tobacco leaves,and visualization of accumulated oil in leaves by Nile red staining.Compared with the mock control leaves,the number of oil drops in the leaves with Pr DGAT1 and Pr PDAT2 was significantly increased.The fatty acids in tobacco leaves were quantified using gas chromatography detection.The results showed that compared with the mock control leaves,the relative amount of C18:2 and C18:3 of the leaves expressing Pr DGAT1 transiently increased,while the relative amount of C18:0 and C18:0decreased.The relative amount of C18:0 and C18:3 of tobacco leaves with transient expression of Pr DGAT2 increased;the relative amount of C18:0 and C18:0 of tobacco leaves with transient expression of Pr PDAT1-1 increased,while the relative amount of C18:2 decreased;The relative amount of C18:1 and C18:3 in transiently expressed Pr PDAT2 tobacco leaves increased.Among them,Pr PDAT2 has the most significant effect on increasing the relative amount of C18:3.4.In order to reverse verify the function of Pr DGAT1,Pr DGAT2,Pr PDAT1-1,Pr PDAT1-2,and Pr PDAT2 in the accumulation of α-linolenic acid on tree peony,we established a VIGS system for tree peony seedlings,which transiently transformed the TRV2-GFP vector with the target genes into tree peony leaves by vacuum-infiltration.Under the UV light,the transformed tree peony leaves were showed obvious green fluorescence,and the green fluorescence was detected under a laser confocal microscope.Transcripts of TRV1 and TRV2-GFP were also detected by PCR,and the expression levels of five acyltransferase genes significantly decreased by q RT-PCR.The fatty acids in tree peony leaves were quantified using gas chromatography detection.The results showed that the relative amount of α-linolenic acid(C18: 3)in the leaves of silence Pr DGAT1 was significantly decreased;the relative amount of oleic acid(C18: 1)in the leaves of silence Pr PDAT1-2 was significantly decreased,while the relative amount of α-linolenic acid(C18:3)significantly increased;the relative amount of α-linolenic acid(C18: 3)in the leaves of silence Pr PDAT2 was significantly decreased,while the relative amount of oleic acid(C18:1)was increased significantly.Based on the above experiment results,it can be concluded that the high level ofα-linolenic acid of tree peony seeds is directly related to the preference of Pr DGAT1 and Pr PDAT2 for α-linolenic acid.Pr DGAT1 and Pr PDAT2 involved an important promoting role in α-linolenic acid accumulation of tree peony seeds.