Cloning And Functional Analysis Of Diacylglycerol Acyltransferase 2 Gene(ElDGAT2) In Euphorbia Lathyris

Euphorbia lathyris is a new type of excellent energy plant with development potential.The accumulation of oil in seeds is up to 60%.Among them,30%-40% of the compounds are similar to the hydrocarbons in petroleum,and they are high-quality raw materials for the production of biodiesel.The main fatty acids in the oil are C16 and C18,especially the content of monounsaturated oleic acid is as high as 83%.Acyl Co A: Diacylglycerol Acyltransferase 2(DGAT2)catalyzes the formation of triacylglycerol(TAG)from diacylglycerol(DAG),which is one of the key enzymes in plant seed oil synthesis.So far,the DGAT2 gene was cloned from plant such as Glycine max,Perilla frutescens,Arabidopsis thaliana,and their functional studies were performed.However,there are few research dates studies on the mechanism of oil synthesis in the seeds and DGAT2 gene of Euphorbia lathyris,although this is particularly important for the sustainable development of high-quality oil products of characteristic oil plants in Euphorbia lathyris.In this stuty,a c DNA that encoding diacylglycerol acyltransferase 2 was isolated and cloned from the developing seed of Euphorbia lathyris(El DGAT2),and The expression profile of El DGAT2 in different organs were investigated.The yeast expression vector and component plant expression vector of were constructed,through the complementary function experiment of yeast and agrobacterium-mediated transient expression in Nicotiana benthamiana,and the total oil content and fatty acid content of transgenic yeast and tobacco leaves were detected,in order to analyze the biological function of El DGAT2 gene.It provides new knowledge for further analysis of the mechanism of oil synthesis and high level accumulation of oleic acid in the seeds of Euphorbia lathyris.El DGAT2 gene can also be used for genetic engineering improvement of plant oil and its quality.The main results are as follows:1.The transcripts of El DGAT2 were identified by screening the Euphorbia lathyris transcriptome database generated previously in our lab,The expression analysis results showed that El DGAT2 gene was expressed in roots,stems,leaves,flowers and seeds,which was significantly higher in seeds than in other organs,the highest expression level in the middle of seed development(30 d days after flowering),and it is the period of rapid synthesis and accumulation of oil,and it reached 12.83 times than that of the leaf.2.The c DNA of El DGAT2 gene was cloned by high-fidelity RT-PCR.The physicochemical properties,high-order structure,subcellular localization and phylogenetic tree of the El DGAT2 protein were analyzed by bioinformatics tools.The full-length El DGAT2 c DNA was 1939 bp and the ORF was 984 bp,encoded327 amino acids.Secondary structure analysis indicated that the major structural elements were alpha-helix(37.00%)and random coil(34.25%).The results of multiple sequence alignment showed that El DGAT2 protein had seven typical conserved domains of DGAT2 enzyme protein family.The phylogenetic tree based on DGAT2 protein showed that the El DGAT2 from Euphorbia lathyris had closer relationship with other DGAT2 s from Euphorbiaceae,including Ricinus communis,Vernicia fordii and Jatropha curcas.3.The yeast expression vector p YES2.0-El DGAT2 was successfully constructed and transferred into yeast.TLC analysis and Nile Red staining were carried out on the transgenic yeast,and the total oil content and fatty acid content were analyzed.The results showed that the oil body was detected in the transgenic defective yeast,and El DGAT2 gene restored TAG synthesis ability of defective yeast H1246,indicating that the proteins encoded by El DGAT2 genes had the DGAT enzyme activity.And the oil content in the transgenic yeast of El DGAT2 gene were increased by 8.65 %,the fatty acids of 16:0,18:1 increased,This indicates that El DGAT2 gene can increase the total lipid content and may have substrate selective for oleic acid.4.The plant constitutive expression vector p CAMBIA1303-El DGAT2 was constructed successfully,and the function of El DGAT2 gene was identified by agrobacterium mediated transient expression on Nicotiana benthamiana.Identification of RNA levels in transgenic tobacco leaves shows that El DGAT2 gene was successfully transferred into tobacco leaves and expressed effectively.The total oil content,fatty acid composition and content,protein and starch content of transgenic tobacco leaves were detected.The results showed that the total oil content of tobacco leaves expressed by El DGAT2 increased by 1.59 %,the saturated fatty acids(16:0,18:0)decreased,and the oleic acid(18:1)and unsaturated fatty acids increased.,indicating that El DGAT2 encodes a catalytically active DGAT2 enzyme protein,heterologous expression of El DGAT2 can increase the synthesis and accumulation of total oil and unsaturated fatty acids in host organs,and El DGAT2 has substrate preference for unsaturated fatty acids such as oleic acid.Moreover,the content of starch and protein in transgenic tobacco leaves decreased.In brief,this study is the first time to identify and clone the gene of El DGAT2.It is the second enzyme protein with DGAT activity after El DGAT1,which can significantly promote the accumulation of oil in host organs.It provides a scientific reference for the analysis oil synthesis molecular mechanism of Euphorbia lathyris,and the development and utilization of oil crops.