| Salvia miltiorrhiza Bunge is a well-known medicinal plant in the Labiatae family.Its dry roots or rhizomes(called 'danshen')are used for the treatment of cardiovascular,cerebrovascular,cancer and inflammatory diseases.In recent years,S.miltiorrhiza is widely accepted as "medicinal herbal model" for investigating the plant secondary-metabolite biosynthesis.Accompanied by the growing demand of S.miltiorrhiza,to improve the content of the active ingredients and cultivate new varieties with high quality have become the' most urgent and key problems in the development of Salvia resources.The active constituents of S.miltiorrhiza can be divided into two groups:lipid-soluble tanshinones,such as tanshinone IIA and cryptotanshinone;and the other is water-soluble phenolic acids,including caffeic acid,danshensu(3,4-dihydroxyphenyllactic acid),rosmarinic acid and salvianolic acid B.The phenolic acids from S.miltiorrhiza,especially rosmarinic acid and salvianolic acid B,.are drawing considerable attention in recent years because they are the main components in water-decoction,which is the major form administered to patients in clinical medication of China.At present,the backbone pathway of phenolic acid biosynthesis was proposed in S.miltiorrhiza.Most genes encoding enzymes in phenolic acid biosynthesis pathway were cloned and their functions were studied preliminary.Howeveer,the regulation genes(e.g.,transcription factors)of these enzymes are still unclear.The reports for the regulation pattern and molecular mechanism of these active constituent biosynthesis and accumulation are also lacking.In view of the above-mentioned facts,we analyzed the global change of gene expression and metabolic flux distribution of S.miltiorrhiza under methyl jasmonate treatment based on transcriptomics and metabolomic strategy,and screened the key regulation genes affecting the phenolic acid synthesis.Furthermore,we try to enhance the contents of water-soluble active constituents of S.miltiorrhiza adopting gene transformation and combinatorial regulation methods,and gain insight into the regulation pattern and molecular mechanism of the active constituent biosynthesis and accumulation in S.miltiorrhiza.The main results and conclusions are as follows:1.Establishing a metabolomic strategy of S,miltiorrhiza based on chromatography-mass spectrometry hyphenated techniquesAnalytical methods of GC-MS and LC-MS for metabolomic study of S.miltiorrhiza were established for the first time.With condition optimization,methodology examination revealed a good repeatability,high sensibility and high-throughput characters of the proposed method.A total of 132 primary metabolites and 150 secondary metabolites were detected in S.miltiorrhiza roots,including organic acids,amino acids,fatty acids,oligosaccharides,phenolic acids,and tanshinones.2.Analyzing the global change of gene expression and metabolic profile of S.miltiorrhiza under methyl jasmonate treatmentBased on the digital gene expression profiling(DGE)and metabolomic strategy,we analyzed the global change of gene expression and metabolic profile of S.miltiorrhiza under methyl jasmonate treatment.A total of 45 symbolic metabolites with significant change were extracted and identified,such as the intermediates in TCA cycle,amino acids,fatty acids,oligosaccharides,phenolic acids,and tanshinones.We also screen out 2131 differentially expressed Unigenes,among which 45 were in secondary metabolic pathway and 42 were transcription factors.The results indicated that methyl jasmonate is an important phytohormone that modulates various physiological processes in plants.Furthermore,a "gene-to-metabolite" network was constructed according correlation analysis.By analyzing the relation between gene expression and metabolite accumulation,we speculate that a new regulation pattern,[PAPl regulation?key genes(like PAL)expression?salvianolic acid B accumulation]exists in salvianolic acid B biosynthesis.3.Establishing new transgenic S.miltiorrhiza with high antioxidant capacity and phenolic acid contentThe Arabidopsis PAPl transcription factor was introduced into S.miltiorrhiza genome,and 4 transgenic lines had obvious overexpression.The highest expression of At PAPl was detected in line PAP1-14.The roots of line PAP1-14 contained approximately 73.27 ± 1.68 mg/g salvianolic acid B,which represented 2.25-fold greater accumulation than in control line.Total phenolics,total flavonoids,anthocyanin,and lignin were also significantly enhanced,and the antioxidant capacity improved up to 3-fold in transgenic plants.AtPAPl activated a broader spectrum of genes,such as PAL,C4H,4CL,RAS and 7TA in the phenylpropanoid pathway.It is effective in enriching the formation of phenolic acids in S.miltiorrhiza,but no significant differences were observed for lipid-soluble tanshinones in transgenic plants.Besides,AtPAPlinduced CCR and COMT high transcription and enhanced the metabolic flux in lignin pathway.4.Constructing a new multi-gene coexpression-cosupression vector system,and further increasing the salvianolic acid B content in transgenic S.miltiorrhizaBased on the new multi-gene coexpression-cosupression vector system,we regulated the phenolics production and improved salvianolic acid B content up to 3-fold in transgenic S.miltiorrhiza by simultaneous over-expression of PAPl transcription factor and cosuppression of CCR-COMT genes.Total phenolics,total flavonoids,anthocyanin,and antioxidant capacity were also enhanced,but the contents of total lignins and S lignin monomer were significantly decreased in transgenic lines PCC-6 and PCC-7.The dynamic change of metabolites in PCC-7 was as same as PAP1-14 by metabolomic analysis.The results of gene expression profiling indicated that most genes in phenolic acid biosynthesis pathway,such as PAL,4CL,RAS,TAT and HPPR,were obviously increased as a result of AtP AP1 overexpression.Owing to the cosuppression of CCR and COMT,almost all genes in lignin pathway were suppressed,and the metabolic flux partial to phenolic biosynthesis.This research is of great significance for the further studies of the secondary metabolite regulation and molecular breeding of S.miltiorrhiza,and provides a promising strategy for genetic engineering of other medicinal plants. |
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