Study On The Lignan And Flavonoid Biosynthetic Pathway In Schisandra Chinensis By Transcriptomics And Metabolomics

SCHISANDRAE CHINENSIS FRUCTUS,also called "North Schisandra",is the dried and mature fruit of Schisandra chinensis(Turcz.)Baill,which is a common traditional Chinese medicine.S.chinensis has the functions of astringency,invigorating Qi,promoting body fluid,tonifying the kidney and tranquilizing the heart.It has important medicinal and economic value and is often used for prolonged coughing,asthenia and asthmatic,spermatorrhea,frequent enuresis and frequent urination,chromic diarrhea,spontaneous perspiration and night sweating,fluid injury and thirst,internal heat and thirst,palpitation and insomnia.At present,most researches of S.chinensis have been focused on chemical composition analysis,pharmacological activity screening as well as quality evaluation of medicinal materials.Although researches on the cultivation,breeding and biological characteristics of S.chinensis have been successively carried out,the analysis of the synthetic pathway of secondary metabolites in S.chinensis and the discovery of key enzyme genes have not yet been conducted.Without the related information,the study of the secondary metabolism regulation and molecular assisted breeding for improving the quality of S.chinensis cannot be carried out effectively.In the present study,we used widely-targeted metabolomics and high-throughput sequencing to analyze the fruits of S.chinensis in three stages of fruit ripening,i.e.green fruit stage,light red fruit stage and deep red fruit stage,to illustrate the transcriptional regulation and metabolic changes during fruit ripening,and to explore the synthesis pathway of lignans and flavonoids in S.chinensis by transcriptional metabolism correlation analysis.These results may lay a foundation for the analysis of the quality formation mechanism of S.chinensis.The main results are as follows:(1)Transcriptome analysis of the three stages of S.chinensis fruit ripening was performed using high-throughput sequencing technology.A total of 83.29 Gb clean data were obtained,with an average clean data of 8.46 Gb for each sample,and Q30 base percentage of 95.36%.A total of 81 095 unigenes were obtained after assembly,with an average length of 1 364 bp for each gene,of which 18 489 unigenes were longer than 1 kb.The functions of unigenes were annotated,by comparing with NR,Swiss-Prot,KEGG,COG,KOG,GO,and Pfam databases,and a total of 41 824 unigene annotation results were obtained.By comparing the differenttranscriptomes in different fruit stages,we found that a total of 413 genes were differentially expressed,of which 196 were up-regulated and 217 were down-regulated.Compared with the light red fruit stage and the dark red fruit stage,a total of 1 144 genes were differentially expressed,of which 500 genes were up-regulated and 644 genes were down-regulated.(2)Using widely-targeted metabolomics,a total of 736 compounds were identified in fruits of S.chinensis in different stages of fruit ripening.According to the types of compounds,these compounds can be divided into 32 categories,the most of which are 89 amino acids and their derivatives(12.09%),72 organic acids and their derivatives(9.78%),61 flavonoids(8.28%),and 54 nucleosides acids and their derivatives(7.33%),and the number of other types of ingredients was less than 50.Using multivariate statistical analysis,a PLS-DA model was established to analyze the specificity between tissues in each stage.Finally,a total of 15 differential metabolites were identified as the main factors to distinguish between different stages(VIP> 1,p<0.05).(3)The genes and metabolites related to lignan synthesis in the transcriptome and metabolome of S.chinensis were thoroughly explored,the lignan biosynthetic pathway was annotated,and the dynamic changes of lignan components during fruit ripening were analyzed.The transcriptional regulation of lignan synthesis pathway was analyzed from the aspects of transcription and metabolism.The association between lignan traits and genes was constructed using WGCNA analysis,and a total of 84 lignan pathway genes and 22 metabolites were annotated.In "green fruit-light red fruit" stage,a total of 43 lignan pathway genes were related to changes in metabolites,and in "light fruit-dark red fruit" stage,a total of 32 lignan pathway genes were related to metabolite changes.Twenty-three genes participated in both stages and are related to changes in metabolites.On this basis,finally,17 genes involved in lignan synthesis were screened by combining with WGCNA results.(4)The biosynthetic pathway of flavonoids was annotated.The dynamic accumulation pattern of flavonoid pathway compounds was obtained through analyzing the changes of flavonoids and the transcriptional regulation of pathways from the aspects of transcription and metabolism,and the gene expression patterns annotated to the flavonoid pathway were also explored.The association between flavonoids and genes was constructed using WGCNA analysis,and a total of 32 flavonoid pathway genes and 21 metabolites were annotated.In "green fruit-light red fruit" stage,26 flavonoid pathway genes were related to changes in metabolites.In "light red fruit-dark red fruit" stage,20 flavonoid pathway genes were related to changes in metabolites.A total of 17 genes participated in both stages and were related tochanges in metabolites.On this basis,8 important genes involved in lignan synthesis were screened by combining with WGCNA results.