| Objective:(1)HPLC was used to determine the contents of six effective components in G.jasminoides fruit at different development stages,and the variation law was clarified.(2)Transcriptomics and metabolomics were used to study the fruit of G.jasminoides,and the regulatory mechanism of transcription and metabolism in the development and maturation of G.jasminoides fruit was analyzed.Through the combined analysis of omics,the key genes in the biosynthesis pathway of iridoids and crocin components in the fruit were identified,which provided a theoretical basis for the research on the biosynthesis of effective components in G.jasminoides Ellis and molecular assisted breeding.Method:(1)Content determinationAccording to the color change characteristics of G.jasminoides fruit development,through the change process of green,yellow-green,yellow,yellow-red and red,G.jasminoides fruit was divided into five development stages:young fruit,green fruit,yellow fruit,yellow-red fruit and red fruit.G.jasminoides fruit in five development stages was collected,and the contents of six effective components including geniposide,genipinic acid,genipin-1-β-gentiobiglycoside,crocin Ⅰ,crocin Ⅱ and chlorogenic acid were determined by HPLC.(2)Transcriptomic analysisThe high-throughput sequencing technology was used to study the fruit of G.jasminoides at different development stages.The genes in each sample were compared and functionally annotated using the public database.The differential expression of the obtained genes was analyzed using DESeq2.The expression trend of the genes was analyzed based on the STEM sequential clustering algorithm.The GO and KEGG databases were used for functional enrichment analysis of the differentially expressed genes.(3)Metabolomics analysisThe non-target metabolomics technology was used to study the changes in the types,numbers and relative contents of metabolites in the fruits of G.jasminoides at different development stages.The differential metabolites were screened by multivariate statistics and one-dimensional statistical analysis.The hierarchical cluster analysis revealed the changes of differential metabolites in different fruit stages.The KEGG database was used to annotate the functional pathways and enrich the differential metabolites in the fruits of G.jasminoides.(4)Joint analysisThe differential expression genes and metabolites involved in the biosynthetic pathways of iridoids and crocin components in G.jasminoides Ellis were found by combined analysis of transcriptomic and metabolomic data.The biosynthetic pathways of the two components in the development of G.jasminoides fruit were analyzed from the perspectives of transcription and metabolism.Based on Spearman correlation analysis,the correlation network between differentially expressed genes and metabolites was constructed,and the functional genes related to iridoids and crocins in G.jasminoides Ellis were further excavated.The STRING database was used to construct the protein interaction network for the genes involved in the biosynthesis pathways of the two components in G.jasminoides fruit.Cytoscape software was used to visualize the network.Result:(1)The content determination results showed that in the fruits of G.jasminoides at different developmental stages,the content and variation of different effective components were different.The content of genipin-1-β-gentiobiglycoside was the lowest in the young fruit stage of G.jasminoides,and with the development of G.jasminoides fruit,the content decreased gradually.It reached its peak at the yellow-red fruit stage,and then decreased slightly at the red fruit stage,showing a trend of first rising and then decreasing;while the crocus anthosides were not synthesized at the early stage of fruit development of G.jasminoides,and their content increased with the maturity of the fruit.The trend of increasing gradually reached the peak in the red fruit stage.(2)Transcriptome sequencing technology was used to study 15 samples of fruits at five stages of development and maturation of G.jasminoides.A total of 103.63 Gb sequencing data were obtained,and 77,310 genes were obtained after assembly.The difference analysis of G.jasminoides fruits at different development stages was carried out,and a total of 17,284 differentially expressed genes were obtained.Among them,the number of differentially expressed genes in young fruit stage and red fruit stage of G.jasminoides was the highest,which was 9375,and the number of differentially expressed genes in yellow fruit stage and yellow-red fruit stage was the lowest,which was 1068.GO and KEGG enrichment analysis of differentially expressed genes was carried out based on time series analysis,and it was found that the fruit of G.jasminoides required the catalysis and regulation of various enzymes in the growth and development process,and also involved the biosynthesis pathways of various secondary metabolites,including phenylpropanoid biosynthesis,carotenoid biosynthesis,and terpenoid skeleton biosynthesis and other pathways.(3)Non-target metabolomics was used to study the metabolites in the fruits of G.jasminoides at different development stages.A total of 743 metabolites were identified.According to the types of plant secondary metabolites in the KEGG database,they can be divided into six categories.Among them,flavonoids,terpenoids and phenylpropanoids have a large number of metabolites,which are 28,26 and 21,respectively.A total of 352 differential metabolites were screened by multivariate statistical analysis and T test,including 238 up-regulated metabolites and 114 down-regulated metabolites.KEGG function annotation and enrichment analysis were carried out.It was found that amino acid metabolism,biosynthesis of secondary metabolites,ABC transporters and carbon metabolism are more prominent pathways in the development of G.jasminoides fruit.(4)Through the combined analysis of transcriptomics and metabolomics,the biosynthetic pathway of iridoids in G.jasminoides Ellis was constructed,and 14 enzymes(ACAT,HMGCS,HMGCR,MVK,PMVK,MVD,DXS,DXR,ispD,ispE,ispH,IDI,GPPS,10HGO)and 8 metabolites.Identification of 16 enzymes(DXS,DXR,ispD,ispE,ispH,GPPS,IDI,crtB,PDS,Z-ISO,ZDS,crtISO,lcyB,CCD4a)encoded by 27 genes in the crocin component biosynthetic pathway,UGT75L6,UGT94E5)and 6 metabolites.The dynamic analysis of the above genes and metabolites during the development and ripening of G.jasminoides fruit was carried out.The results of differentially expressed genes and metabolites correlation network showed that there were 211 genes highly related to iridoids and 153 genes highly related to crocin in G.jasminoides fruit.(5)Based on the STRING database,the protein network interaction analysis of the genes involved in the synthesis of iridoids and crocin in G.jasminoides fruit was carried out.According to the degree of connectivity(Degree)of each gene node in the interaction network,two types of Key genes in component synthesis pathways.The analysis showed that the key gene in the synthetic pathway of iridoids in G.jasminoides Ellis was HMGCR,and the key genes in the synthetic pathway of crocins were DXS,DXR,PDS3 and PSY.Conclusion:(1)The contents of six effective components in G.jasminoides fruit at different development stages were determined,and the change rule was clarified.There were some differences in the content and variation of different effective components.They were affected by the fruit development stage.The iridoids components represented by gemposide showed a decreasing trend,while the crocin components showed an increasing trend.The two components showed opposite accumulation patterns in G.jasminoides fruit.(2)Based on transcriptome and metabolomics,the fruit of G.jasminoides at five developmental stages was analyzed,and 14 enzymes encoded by 8 metabolites and 29 genes involved in the biosynthesis pathway of iridoids were identified,among which HMGCR was the key regulatory gene.In the biosynthetic pathway of crocins,16 enzymes encoded by 6 metabolites and 27 genes were identified,among which DXS,DXR,PDS3 and PSY played key regulatory roles.These key genes may play an important regulatory role in the biosynthesis of G.jasminoides Ellis iridoids and crocins.In this study,the biosynthesis pathways of iridoids and crocins in G.jasminoides fruit were analyzed,and the key genes for the biosynthesis of two effective components were identified,which was beneficial to the further development and utilization of G.jasminoides Ellis,and provided theoretical basis for genetic research and molecular assisted breeding of G.jasminoides Ellis. |
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