| Salvia miltiorrhiza Bunge, a perennial herbaceous plant, belong to the Labiatae family. Its dry roots or rhizomes (called’danshen’) are used as an important Chinese traditional medicine with a long history. It bear various properties such as protecting cardiovascular system, preventing thrombosis, promoting the repair and regeneration of tissue and so on. Studies have reported that the active constituents of S.miltiorrhiza can be divided into two groups:lipid-soluble tanshinones and water-soluble phenolic acids.The later is the main medicinal components and gained wider attention.It was found that phenolic acid compounds synthesized through two metabolic pathways of phenylpropanoid and tyrosine in Salvia miltiorrhiza, and rosmaric acid is the core precursor of salvianolic acid B. At present the researches of functional gene have been progressed, at the same time the key enzyme genes of phenolic acids biosynthesis have been cloned and wildy regulated by transcription factors. We noticed that the enzymes genes of phenolic acid metabolilic pathways changed after overexpressing exogenous MYB gene (Arabidopsis PAP1,PAP2) in Salvia miltiorrhiza, whether the endogenous transcription factors have the same function is also worthy to study. SmPAPl (GenBank:GU218694.2) is an gene encoding for R2R3MYB transcription factors of Salvia miltiorrhiza and the gene sequence has a high degree of similarity with AtPAP1. In order to explore the potential regulatory function of the SmPAP1in phenolic acid components synthesis of Salvia miltiorrhiza, we constructed an artificial miRNA (amiRNA) plant expression vector for specificly silencing SmPAPl gene, followed by transformation into Salvia miltiorrhiza, and further analyze effect of down-regulation of SmPAP1gene expression on the phenolic acids synthesis in Salvia miltiorrhiza.This research aims to investigate the regulation mode of endogenous MYB transcription factor and phenolic compounds of Salvia miltiorrhiza, and provide a new direction and ideas for other secondary metabolites researches in medicinal plants.The main results were as follows:1. Based on the noconservative region of the SmPAP1, we designed an amiRNA to specifically slience SmPAPl with the WMD (Web microRNA designer) online.Then substituted this amiRNA/amiRNA*for the miRNA319/miRNA319*duplex of Arabidopsis thaliana miR319precursor using overlapping PCR for the amiRNA construct formation.2. Using realtime quantitative PCR technology, we analyzed the expression level of SmPAPl of Salvia miltiorrhiza roots, stems and leaves tissues. It showed that SmPAP1express to a different extent in the examined tissues of S.miltiorrhiza, where in highly in leaves, moderate in stem, and the lowest in roots.3. Using Agrobacterium-mediated transformation method and detected by DNA and RNA test, we generated8transgenic positive lines of amiRNA-SmPAP1. Semi-quantitative RT-PCR and realtime quantitative PCR characterize the expression of SmPAPl and amiRNA in transformed lines. Results showed that SmPAP1expression was obvious lower in transformed lines compared to control lines, and the silencing ratio range from25%to97%. Besides, amiRNA expression was obvious higher than control lines that proved the mature miRNA were successfully expressed and silenced target gene in transformed lines. On the other hand, in transformed plant, SmPAP1expressionin roots, stems and leaves tissue were similar to that of wild type plant.4. Analysis results showed that the contents of total phenolic, total flavonoids and anthocyanin were reduced in45d plants transformed lines compared with that of wild type plant. The data in more detailed as follows:compared with control, total phenolic, total flavonoids and anthocyanin contents of4#decreased by61.56%、64.05%and49.47%. And that of9#and24#were48.42%,56.88%and36.81%as well as73.68%,77.57%and38.35%, respectively.5. Analysis results showed that the content of salvianolic acid B was reduced in45d plants transformed lines by HPLC, the results showed that:compared with control, salvianolic acid B contenes of4#and9#decreased by44.94%and50.72%, respec-ively。6. To further explore the correlation relationship between the SmPAPl gene expression level and the phenolic acid biosynthetic pathway, realtime PCR was used to analyze mRNA aboundance of the10key enzyme genes involved in phenolic acids synthetic. The results showed that:the expression level of PAL、C4H、4CL1、RAS genes of core phenylpropanoid pathway and TAT, RAS genes of tyrosine metabolic pathways were obviously reduced compared with contol. That CHS, FLS genes of flavonoid pathway and the HCT, COMT gene of lignin pathway were also, to a different extent, decreaed in transformed sliencing lines. 7. The phenotypic observations of SmPAP1gene silencing lines showed, after45d growing in soil,the transgenic and control lines shows great differents:because the key enzymes of phenolic acids, lignin and flavonoids pathway have reducted, so it is insufficient of the lignin, phenolic acids and flavonoids content. It is the reason that the taproots single and small, the major are fibrous roots and looks like pink.The whole plant weaker than wild Salvia miltiorrhiza.Conclusion:in this study, we constructed artificial miRNA to specifically silence the SmPAP1gene of S.miltiorrhiza and gained transgenic plants. Results showed that the SmPAP1transcription factor played an important role in the regulation of phenolic acids synthesis and normal growth through dominating expression of the relevant key enzyme genes in the pathyway. The results will contribute to the research of transcription factor complex and provide a new idea to improve the secondary metabolites of Salvia miltiorrhiza. |
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