Genetic Engineering Of Lipid Metabolism In Biofuel Plant Jatropha Curcas

Jatropha curcas L.is considered as one of the most promising oilseed plants.Its seed oil content is up to 40%and rich in oleic acid.As a non-edible oilseed crop,J.curcas naturally does not compete with grain for land and can adapt to the poor environment.It is an energy plant resource with great potentials.However,due to the low oil production of J.curcas,it is still a crop with low economic value,and it is difficult to commercialize and plant on a large scale.To improve the economic value of J.curcas,this research is mainly devoted to increasing the oil production of J.curcas and improving the quality of the oil so that it can better meet the market demand.In the lipid biosynthesis pathway of plants,diacylglycerol acyltransferase(DGAT)is the only enzyme catalyzing the acylation of sn-1,2-diacylglycerol(DAG)to form triacylglycerol(TAG),and is a rate-limiting enzyme of the Kennedy pathway.The main DGAT enzymes in J.curcas are JcDGAT1 and JcDGAT2,which have previously been verified in heterologous systems.In this study,the functions of JcDGAT1 and JcDGAT2 were verified in J.curcas with the aim of improving the oil production of J.curcas and deepening the understanding of their roles in J.curcas.The results showed that JcDGAT1 and JcDGAT2 had similar expression patterns and were found to be predominantly expressed at the late stages during J.curcas seed development,in which large amounts of oil accumulated.We overexpressed the JcDGAT1 or JcDGAT2 under the control of the CaMV35S promoter in J.curcas.As expected,the oil content of 35S:JcDGAT1 and 35S:JcDGAT2 transgenic J.curcas seeds was significantly increased,reaching 53.7%and 55.7%of the dry weight of seed kernels,respectively,which was 25%and 29.6%higher than that of the control plants.The increase in seed oil content was accompanied by decreases in the contents of protein and soluble sugars in seeds.The content of TAG in transgenic leaf was 2-4 times that of control leaves.Additionally,the percentage of fatty acids in the lipid of seeds and leaf altered significantly.Moreover,by analysis of the fatty acid(FA)profiles,we found that JcDGAT1 and JcDGAT1 had the same substrate specificity with preferences for linoleic acid(C18:2)in seed TAG,and hexadecanoic acid(C16:0),octadecanoic acid(C18:0),and oleic acid(C18:1)in leaf TAG.Therefore,our study confirms the important role of JcDGAT1 and JcDGAT2 in regulating oil production in J.curcas.The synthesis and accumulation of oil in non-seed tissues and organs of plants have attracted much attention in recent years,and many genes related to the lipid metabolism pathway have been applied in the genetic engineering research of non-seed tissues such as leaf and tuber.In order to improve the production of J.curcas oil,we hope to increase the oil production in the leaf which was with large biomass.In this study,Arabidopsis WRINKLED1(AtWRI1),Arabidopsis diacylglycerol acyltransferase gene(AtDGAT1),and Sesame olesome protein gene(SiOleosin)were co-expressed in J.curcas to improve the quantity and quality of leaf oil.The results showed that the TAG content in transgenic leaf increased to 343 ug/100 mg,which was 10 folds the content in the wild-type leaf,which was accompanied by the decreased in the contents of the soluble sugar and starch.In addition,the protein content increased to 4 times the total protein of wild-type leaf.Consistent with the quantitative analysis results,the neutral lipid droplets were observed by laser confocal microscope,which showed that the density of lipid droplets in the transgenic mesophyll cells increased significantly and became denser.The results from laser confocal microscopy showed that the neutral lipid droplets in transgenic mesophyll cells became denser.This is also consistent with our quantitative results.The fatty acid compositions of TAG in transgenic leaves altered significantly.The percentage of palmitic acid(C16:0),oleic acid(C1 8:1),and linoleic acid(C18:2)in TAG molecules increased significantly,while the percentage of linolenic acid(C18:3)decreased.It indicates that the saturated degree of fatty acid component is higher and the stability became well.To enhance the understanding of the lipid metabolism in J.curcas leaf,lipid metabolomics integrated with the transcriptomics analysis was performed in leaf from the wild-type and the pOIL040 transgenic line.Transcriptome data showed that many genes involved in the glycolysis,fatty acid synthesis,glycerolipid,and glycerophospholipid metabolic pathway were regulated in transgenic leaves.We verified the expression level of these genes by qRT-PCR.The lipid metabolomics data showed that the absolute content of free fatty acid(FFA),diacylglycerol(DAG),phosphatidylcholine(PC),monogalactosyldiacylglycerol(MGDG),and digalactosyldiacylglycerol(DGDG)were also increased significantly.Besides,the percentage of lipids in the leaf altered.The percentage of TAG in all lipids in the leaf increased significantly,while the percentage of polar lipids such as PC,PG,and DGDG decreased,only MGDG increased slightly.The proportion of the molecular type of each lipid changed compared with the wild-type.The results from the lipid metabolomics and the transcriptomics illustrated that co-expressed the AtWRI1,AtDGAT1 and SiOleosin made a significant impact on the leaf lipids metabolism pathway.These results are of great significance for us to optimize the lipid metabolism pathway in J.curcas leaf.