Molecular Regulation Mechanism Of Triacylglycerol Synthesis Triacylglycerol Synthesis Rate-limiting Enzyme DGAT1 In The Seed Of Tree Peony

Tree peony(Paeonia)is a perennial garden plant originated in China.Its fruit seeds contain various types of unsaturated fatty acids,among which α-linolenic acid has a high proportion and high nutritional value.Peony seed oil is rich in more than 40 % α-linolenic acid.The selective transfer of α-linolenic acid on the glycerol skeleton can affect the composition and accumulation of vegetable oils,but its mechanism has not been elucidated.Previous studies have shown that the sn-3 position of peony seed oil is the main binding site of α-linolenic acid.Acyltransferase DGAT,as a rate-limiting enzyme for fatty acid transfer at sn-3 position of plant oil,participates in the efficient accumulation of peony seed oil.In this study,the PrDGAT1 gene promoter was cloned and its characteristics were analyzed based on the cloning and functional verification of DGAT1 gene in P.suffruticosa.The transcription factors that bind to the PrDGAT1 gene promoter were further selected by yeast single hybridization and double luciferase reporter system.The selected transcription factors were transiently transformed into tobacco,over-expressed in TRV-VIGS and Arabidopsis,and the role of the target transcription factors in the accumulation of α-linolenic acid was verified.Finally,the molecular model of efficient transfer of α-linolenic acid from peony seeds to oil was clarified,which provided theoretical support for the breeding of new varieties with high α-linolenic acid in oil plants.The main research results are as follows :1.The promoter of PrDGAT1 gene was cloned by chromosome walking,and a total length of 1162 bp was obtained.PrDGAT1 pro : GUS fusion expression vector was constructed for stable transformation of Arabidopsis.Results Strong GUS signal was detected in flower of PrDGAT1 promoter.The GUS staining was deepest and most active in anthers.No GUS signal was found in other tissues.We constructed a5 ’-deletion PrDGAT1 pro : GUS fusion expression vector,and infected Nicotiana benthamiana by Agrobacterium.GUS staining showed that the strongest GUS signal was at-310 /-1bp,indicating that the promoter activity was the highest at-310 /-1bp.At the same time,we also found that the promoter activity of PrDGAT1 gene gradually decreased with the increase of promoter.2.Construction of yeast one-hybrid cDNA library of Paeonia suffruticosa seeds.The p His2-PrDGAT1 plasmid was co-transformed with yeast c DNA library.A total of 56 positive yeast strains were obtained from yeast single hybridization library.After sequencing,the selected transcription factor PrGTE7 was verified,and its CDS region was constructed to p GADT7,which was verified by one-on-one yeast single hybridization with p His2-PrDGAT1.The dual luciferase reporter system verified the binding of transcription factor PrGTE7 to the promoter of PrDGAT1.The ratio of LUC and REN in leaves was detected by a multifunctional microplate reader,and the results showed that the ratio of LUC / REN of PrGTE7 + p DGAT1-0800 was 2.2 times that of 35 S + p DGAT1-0800.It indicates that PrGTE7 transcription factor binds to PrDGAT1 promoter and plays a regulatory role.3.The expression profiles of PrGTE7 transcription factor in different developmental stages and different tissues and organs were analyzed by RT-PCR and q RT-PCR.The results showed that the expression of PrGTE7 transcription factor increased gradually in the S1-S5 period of P.yezoensis seeds,and tended to be flat after the S4 period,which was basically consistent with the accumulation trend of α-linolenic acid(C18 : 3)in P.yezoensis seeds.The subcellular localization results showed that PrGTE7 protein was mainly localized in the nucleus and cytoplasm.The transcriptional activity of PrGTE7 protein was analyzed,and the results showed that PrGTE7 gene was a transcription factor with transcriptional activation activity.4.PrGTE7 gene was transiently expressed in Nicotiana benthamiana leaves by constructing a combined expression vector of p RI101-AN and PrGTE7 gene,and the oil accumulation in leaves was visualized by Nile red staining.Compared with the blank control leaves,the oil droplets in the leaves transfected with PrGTE7 gene increased significantly.The fatty acids in tobacco leaves were analyzed by gas chromatography.The results showed that the total oil content in tobacco leaves with transient expression of PrGTE7 gene increased significantly.In terms of fatty acid proportion,the proportion of C18 : 3 increased significantly.The role of PrGTE7 gene in oil accumulation was reversely verified by virus-induced gene silencing(VIGS)on peony.The fatty acid content in the treated peony leaves was determined by gas chromatography.The results showed that the total oil content of gene silencing PrGTE7 leaves was reduced,and the proportions of C16 :0,C18 : 0 and C18 : 1 were significantly reduced.At the same time,q RT-PCR was used to detect other oil-related genes in peony seeds.The results showed that most of the genes were significantly down-regulated.In summary,PrGTE7,the upstream transcription factor of PrDGAT1 gene,was cloned and identified for the first time in Paeonia suffruticosa in this study,indicating that PrGTE7 transcription factor can regulate seed oil synthesis of Paeonia suffruticosa by regulating the expression of PrDGAT1 gene,providing molecular targets for improving seed oil content and quality of Paeonia suffruticosa,and can also be used as breeding practice for improving seed oil quality of other transgenic plants.