Characterization And Functional Analysis Of Diacylglycerol Acyltransferase(DGAT) Gene From Oil Palm (Elaeis Guineensis Jacq.) Mesocarp

Oil palm(Elaeis guineensis Jacq.)is one of the highest yielding oil plants in the world.Because of its high oil content and large oil production per unit area,it is known as the"World Oil King"(Lei Xintao,Cao Hongxing et al.2012).As one of the most characteristic tropical economic crops,its palm oil is mainly derived from the mesocarp of oil palm fruit,storing 90%and 60%(dry weight)oil in its mesocarp and kernel,respectively.It is the type of plant tissue with the highest oil content reported so far.However,research on the accumulation and metabolism of vegetable oils involves almost no type of tissue material other than seed.To gain insights into the oil accumulation mechanism in oil palm,two of the key enzymes involved in triacylglycerol(TAG)biosynthesis,type 1 and 2 diacylglycerol acyltransferase were cloned and characterized its functional activities.The main results are as follows:1.In the second and fourth stages of oil palm fruit development,oil palm type 1 diacylglycerol acyltransferase(EgDGATl)is highly expressed in mesocarp.In the last two periods of oil palm fruit development(fourth and fifth periods),oil palm type 2 diacylglycerol acyltransferase(EgDGAT2)was highly expressed in the mesocarp,while the oil accumulation rate gradually increased during the maturation of oil palm fruits.2.RNA was extracted from oil palm mesocarp,and the full-length EgDGAT2 sequence was obtained by 5'RACE.EgAGDT1 and EgDGAT2 were amplified by PCR and were 1710bp and 999bp in size,respectively.Bio informatics analysis showed that the EgDGAT1 gene has a conserved domain at the C-terminus and belongs to the DGAT family.Correspondingly,EgDGAT2 contains two predicted transmembrane domains,which were adjacent and located near the N-terminus.Moreover,it also possesses conserved and functional motifs of DGAT2.All these characteristics of the protein sequence strongly indicated this protein EgDGAT2 belongs to DGAT2 family,rather than DGAT1.Furthermore,the evolutionary analysis supported this hypothesis as EgDGAT2 clustered with DGAT2 proteins from other plant species,especially AtDGAT2 from Arabidopsis.3.EgDGATl and EgDGAT1 were ligated to the pYES2 vector to construct a yeast expression vector and transferred into the defective yeast H1246 to verify the in vivo activity and function of the EgDGAT2 gene in yeast.The results showed that this gene was able to restore the function of H1246 yeast to restore TAG synthesis and favored the synthesis of palmitic acid(C16:1)and oleic acid(C18:1).4.To further explore the in vivo activity of EgDGAT2 in plants,EgDGAT2 gene was ligated with pCAMBIA1300s-napin vector to construct a plant expression vector and transformed into Arabidopsis through.Three strains of homozygous Arabidopsis thaliana expressing a range gradient were screened by fluorescence quantitative detection.The oil content and fatty acid composition were detected after the seeds were collected.The results show that seed-specific overexpression of EgDGAT2 in Arabidopsis thaliana increased the content of polyunsaturated fatty acids linoleic acid(C18:2)and linolenic acid(C18:3)(each by 6 mol%)in seed TAGs.Correspondingly,the proportion of stearate(C18:0)fatty acids decreases.At the same time,the content of arachidic acid(C20:0)also decreased.In summary,both EgDGAT1 and EgDGAT2 genes belong to the DGAT family,and heterologous expression in the mutant yeast H1246 restores TAG biosynthesis with substrate preference for unsaturated fatty acids palmitic acid(C16:1)and oleic acid(C18:1).In addition,specific overexpression of EgDGAT2 in Arabidopsis seeds also showed a preference for polysaturated fatty acids.These results provide new insights into understanding the in vivo activity of EgDGAT2 from oil palm mesocarp,which will be of importance for metabolic enhancement of unsaturated FAs production.