| Paeonia lactiflora Pall is a perennial herbaceous plant in the genus Paeonia of the Paeoniaceae,known for its high ornamental value.In recent years,with the identification of tree peony seed oil as a new resource food,the oil value of herbaceous peony which belong to the same family and genus as tree peony has also begun to get more attention.Since the current research on oil peony is still in the initial stage,it basically focuses on the variety selection of oil peony,the extraction and physical and chemical properties of seed oil,and the mining of related genes in the fatty acid synthesis pathway,while the molecular mechanism of oil storage of peony medicine is less studied.As an organelle for storing oil,the oil body actively participates in biological functions such as lipid metabolism and lipid storage.The oil content in seeds is related to the oil bodies in seed cells.Therefore,it is of great significance to analyze the molecular mechanism of oil storage of peony seeds from the oil body.In this study,the relationship between seed oil body and oil content was explored through microscopic section observation of seeds,observation of seed oil body stability,and determination of fatty acid composition and content in seeds selected by the research group based on the seeds of HS20(low oil content)and HS37(high oil content)strains with large differences in oil content in the early stage of development.Through transcriptomics thirdand second-generation sequencing technology,the oil membrane protein(OLE)gene family,which plays a very important role in maintaining oil size and stability,was analyzed and screened,and finally the PIOLE4 with the highest expression amount of differentially expressed genes was selected for gene cloning and functional verification to clarify the role of peony PlOLE4 gene in oil storage.The main findings are as follows:(1)The seeds of HS20 and HS37 strains of ’HangShao’ were used as materials for 30,45,60,75 and 90 days after flowering,and the developmental characteristics of seed oil bodies were studied.With the change of seed development period,the lipids in seeds showed a trend of first increasing and then decreasing,and the oil body increased and expanded with the development process,and the volume and quantity of oil in HS37 with high oil content increased slightly compared with HS20 with low oil content.When the NaCl concentration is high,the pH value is acidic and the oil body of peony is more destructive,but the stability of HS37 with high oil content is better than that of HS20 with low oil content,and the temperature has less influence on the structure of the oil body.GC-MS analysis found that there were 5 major fatty acids in the seeds of ’Hangzhou Peony’:palmitic acid,stearic acid,α-linolenic acid,linoleic acid and oleic acid,with the highest content being α-linolenic acid and the lowest being stearic acid;The total fatty acid content of HS37 with high oil content and the content of five major fatty acids were higher than those in HS20 with low oil content.(2)Using the transcriptome sequencing technology of the second generation combined with the third generation,a total of 484931 ROI sequences and 441185 FLNC transcripts were obtained by the third-generation sequencing,and the overall assembly was good.The results of next-generation transcriptome sequencing of 18 independent samples were analyzed as a reference gene bank,and a total of 115.56 Gb of data was obtained,with the data of each clean reads accounting for more than 94.51%of the original data,Through KEGG enrichment pathway analysis,in the three comparison groups of L30D-VS-H30D,L60D-VS-H60D and L90D-VS-H90D.32,20 and 4 differentially expressed genes involved in the biosynthesis of unsaturated fatty acids were found.104,37 and 5 differentially expressed genes involved in fatty acid degradation,respectively.25,22 and 1 differentially expressed genes involved in fatty acid prolongation,respectively.78,33 and 5 differentially expressed genes involved in the metabolism of α-linolenic acid,respectively.There were 11,11 and 0 differentially expressed genes involved in linoleic acid metabolism,77,24 and 3 differentially expressed genes involved in glyceride metabolism,and 105,38 and 2 differentially expressed genes involved in glycerophospholipid metabolism,respectively.At the same time,we identified 10 OLE gene family members,with amino acid numbers between 116-220,molecular masses between 9.21-40.42 kDa,isoelectric points between 5.4-10.45,aliphatic index,protein hydrophobicity,instability coefficient and helical structure.OLE gene family members all have a conserved domain(PF01277),each OLE gene contains 6-9 motifs,while PlOLE4 contains 10 motifs.(3)Based on the results of transcriptome sequencing,the PlOLE4 gene with the highest expression and the most active expression during seed development was screened out as the research object,and PlOLE4 was cloned,and the total length of the nucleotide sequence was 819 bp,the open reading frame was 435 bp,a total of 145 amino acids,and the expression level in HS37 seeds with high oil content was higher than that in its low oil content’HS20.Subcellular localization was observed with the help of tobacco protoplasts and found to be distributed on cell membranes.Through subcellular localization observation using tobacco protoplasts,it was found that they are located in the cytoplasm.After the expression of PlOLE4 in tobacco,the total fatty acid content and main fatty acid content in transgenic tobacco seeds were significantly higher than those in wild-type tobacco seeds,and the functional replenishment experiment of PlOLE4 in Arabidopsis thaliana mutant At2g25140 showed that the total fatty acid content and main fatty acid content in Arabidopsis thaliana seeds after replenishment were higher than those in its mutant seeds,and the content in wild-type Arabidopsis thaliana seeds was equivalent to that in wild-type Arabidopsis thaliana seeds,indicating that PlOLE4 gene has the function of oil storage. |
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