Screening And Functional Identification Of Transcription Factors Related To Regulate Diacylglycerol Acyltransferase (DGAT1) From Elaeis Guineensis Jacq

Oil palm is one of the most efficient oil-producing crops,in which about 90% of the palm oil is accumulated in the mesocarp tissue,which is the type of plant tissue with the highest oil content reported so far.The ratio of saturated and unsaturated fatty acids in palm oil is close to 1:1,and high levels of saturated fatty acids are considered harmful to the human body.Due to the limitations of long fruit development cycle and oil accumulation affected by environmental factors,the basic research on related molecular biology of oil palm is relatively less.In this study,the transcription factors related to the regulation of oil palm diacylglycerol acyltransferase(EgDGAT1)were screened and identified to explore the factors affecting oil accumulation in oil palm mesocarp to improve its yield and quality,and lay a foundation for the study of molecular metabolic mechanism of oil accumulation in oil palm.In this study,the overexpression of EgDGAT1 increase the content of TAG in Arabidopsis seed oil by 42%,and EgDGAT1 plays a key role in controlling oil synthesis.In addition,a transcription factor(named Egb HLH63)that interacts with the promoter of EgDGAT1(pro EgDGAT1)was screened by the yeast one-hybrid system.Studies have shown that Egb HLH63 is located in the nucleus and has transcriptional auto-activation activity.It is a transcription factor belonging to the b HLH family.The fluorescence quantitative results showed that Egb HLH63 and EgDGAT1 have similar dynamic expression trends during oil palm fruit development.The results of yeast one-hybrid,double luciferase reporter gene detection and EMSA in vitro show that Egb HLH63 could bind to pro EgDGAT1 by recognizing E-box motif.The overexpression of Egb HLH63 in transgenic oil palm protoplasts reduced the expression of EgDGAT1 by 11 times compared to wild type.The silencing of Egb HLH63 restored the high expression of EgDGAT1 in transgenic oil palm embryoids,and the content of TAG increased by 73.17%.These results suggest that Egb HLH63 is a transcriptional inhibitor of EgDGTA1,and affects oil accumulation by restricting the transcription and replication of EgDGAT1.To enrich the understanding of role of EgbHLH63 in the regulation of oil metabolism,other functional proteins interacting with Egb HLH63 were screened by the yeast two-hybrid system,including three transcription factors(EgULT1,EgZFP and EgAGL9)and six protein kinases(EgSAPK2,EgOSK1,EgSTY13,EgPFK6,EgKIN10 and EgMAPK3).The results of subcellular localization and yeast autoactivation activity showed that EgULT1 and EgZFP are also located in the nucleus and both have transcriptional autoactivation activities,which is in line with the characteristics of transcription factors.Further results show that EgULT1 and EgZFP could negatively regulate the expression of EgDGAT1 and ultimately affect the accumulation of oil by recognizing B-box and AT-rich motifs and combining with pro EgDGAT1,respectively.The interaction between transcription factors was further verified by yeast two-hybrid,bimolecular fluorescence complementation(Bi FC)and GSTPulldown assay.The results show that both EgULT1 and EgZFP could interact with Egb HLH63,but this interaction does not appear in the combination of EgULT1 and EgZFP.These results deepen our understanding of the mechanism of oil metabolism in the mesocarp tissue of oil palm with the involvement of EgDGAT1,and provide a new research idea for improving the yield and quality of oil palm oil.