| Salvia miltiorrhiza is a plant of the genus Salvia in the family Labiatae.Dried roots and rhizomes of S.miltiorrhiza are known as Danshen which are important medicinal materials in Chinese traditional medicine.Studies have found that S.miltiorrhiza has high levels of genetic diversity,and there are obvious varitations in tanshinones content in different S.miltiorrhiza germplasm.Biosynthesis of tanshinones is a part of life activities of S.miltiorrhiza.However,the research on S.miltiorrhiza at present mainly focuses on the single pathway of tanshinone biosynthesis,while the molecular mechanism of tanshinone biosynthesis and accumulation and the comparative study of different S.miltiorrhiza germplasm at transcriptome level are less.Transcriptomics is a discipline that systematically studies gene expression from the overall transcriptome level,and reveals the relationship between the molecular mechanism of the regulatory network andspecific biological functions and traits.Therefore,in order to study the possible causes of variations among different S.miltiorrhiza germplasm and the molecular mechanism of regulating tanshinone biosynthesis and accumulation,we used UPLC to examine the content of active components in S.miltiorrhiza originated from20 different origins,and screened out S.miltiorrhiza samples from four genetic backgrounds(Zhongjiang in Sichuan,Mile in Yunnan,Luanchuan in Henan,Tongguan in Shaanxi)for transcriptome analysis.The tanshinone content of different S.miltiorrhiza germplasm was determined in the periods of tanshinone accumulation after anthesis.At the same time,Illumina Hi Seq 2500 was used to perform RNA-Seq.After obtainting transcriptome data,gene function annotation,differential expression analysis,and the correlation analysis between the dynamic changes of tanshinones content and differentially expressed genes(DEGs)expression pattern were carried out by using Illumina Hi Seq 2500.The main research results are as follows:(1)Under the same environment condition,the types of secondary metabolites in different S.miltiorrhiza germplasm are similar,but the contents are significantly different.The content of active secondary metabolites in fresh S.miltiorrhiza roots from 20 different producing areas,such as Sichuan,Shandong and Shaanxi,was determined,and a method for simultaneous determination of 12 secondary metabolites in fresh S.miltiorrhiza was established.Ultra.Performance Liquid Chromatography analysis showed that there was no.significant difference in the types of salvianolic acids and tanshinones,but there were significant differences in their contents.LSD analysis of secondary metabolites from different originate producing areas showed that there were significant differences in tanshinone in fresh S.miltiorrhiza.Although the difference of salvianolic acids in S.miltiorrhiza from different originate producing areas is less than that of tanshinones,there is still statistical significance.Through multivariate statistical analysis,six different components which may distinguish the origin of S.miltiorrhiza were screened out,and three components,tanshinone I,tanshinone IIA and dihydrotanshinone I,were identified by comparison of reference substances.Combined with the results of multivariate statistical analysis,S.miltiorrhiza from Sichuan Zhongjiang,Shaanxi Tongguan,Henan Luanchuan and Yunnan Mile,which have obvious differences in tanshinone content,were selected as the materials for the transcriptome analysis.(2)In the tanshinones accumulation stage,the accumulation degree of each component in S.miltiorrhiza is different.In this study,UPLC technique was used to determine the fat-soluble tanshinones in different S.miltiorrhiza germplasm at two stages of tanshinone accumulation after anthesis.The results showed that there were significant differences in six tanshinones in S.miltiorrhiza with different genetic backgrounds during the accumulation periods.The difference is that the accumulation of cryptot.anshinone,tanshinone IIA and tans.hinone I increased greatly,while the accumulation of dihydrotanshinone I,tanshinone IIB and miltirone increased relatively little.(3)Transcriptome sequencing of S.miltiorrhiza roots with four different genetic backgrounds.Transcriptome sequencing of S.miltiorrhiza with four different genetic backgrounds was carried out by using Illumina Hi Seq 2500.After quality control and filtration,we obtained 153.29 Gb Clean datas and the Q30 of each sample was over75.22%.After Trinity assembly,a total of 70,357 Unigene were obtained,.with N50 of1911 bp and average length of 1071 bp.A.total of 40,019(56.88%)Unigene were annotated by four public databases,namely Nr,KEGG,COG/KOG and Swiss Prot.The Get ORF software was used to predict and obtain 39,387 CDS with a total length of32,048,509 bases.All unigenes were screened by MISA software,and 11456 SSRs were identified from 9492 unigenes,with single primitive type as the main type.(4)The accumulation of tanshinone is related to some primary metabolism and signal transduction pathways.The functional classification of DEGs between the two accumulation periods shows that the pathway related to photosynthesis,oxidative phosphorylation,.diterpenoid biosynthesis,.terpenoid backbone.biosynthesis,biosynthesis of secondary metabolites,sesquiterpenoid and triterpenoid biosynthesis are the most differentially regulated processes in these S.miltiorrhiza with different genetic backgrounds in tanshinone accumulation period.In addition,combined with the analysis of the dynamic changes,277 DEGs(including 1 dehydrogenase,3 CYP450 s and 24 transcription factors belonging to 12 transcription factor families)related to the accumulation of tanshinones components were obtained.Furthermore,the KEGG pathway enrichment analysis of these 277 DEGs suggested that there might be an interconnection between the primary metabolic processes,signaling processes and the accumulation of tanshinones components.To sum up,in our research,we analyzed the effects of different genetic backgrounds and accumulation periods on the content of tanshinones from the perspective of macroscopic overall metabolism of plants by using the method of comparative transcriptome,so as to systematically understand the differences in the content of tanshinones of S.miltiorrhiza with different genetic backgrounds in the same environment,as well as the effects of genetic backgrounds,accumulation periods and their interrelationships on active ingredients.The results are useful to illuminate the relationship among tanshinone biosynthesis and accumulation and primary metabolism and signal transduction pathway,and to explore the overall metabolism of plants during tanshinone accumulation.It also provides a new idea for studying the gene regulation mechanism of tanshinone biosynthesis,prepares a ground for elucidating the genetic mechanism of authentic formation of S.miltiorrhiza,and provides a theoretical basis for selecting high-quality provenances of S.miltiorrhiza. |
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