Effects Of Specific Silencing Of Rosmarinic Acid Synthase Gene (SmRAS) On The Synthesis Of Phenolic Acids

Salvia miltiorrhiza Bunge, a perennial medicinal plant, belongs to the Labiatae family and is widely spread in most areas in China. As the main medicinal parts of Salvia miltiorrhiza, the dry roots or rhizomes (called ’danshen’) are used as an important Chinese traditional medicine with a long history. In traditional, S.miltiorrhiza has been widely used for the treatment of cardiovascular diseases, menstrual disorders and the prevention of inflammation. Pharmacological investigations have reported that the active constituents of S.miltiorrhiza can be divided into two main groups:one are lipid-soluble tanshinones, such as tanshinoneⅡA and cryptotanshinone, the other are water-soluble phenolic acids, such as rosmarinic acid and salvianolic acid B. It has been reported that the water-soluble constituents was synthesized through two metabolic pathways deriving from tyrosine and phenylpropanoid respectively in Salvia miltiorrhiza, and the metabolic intermediate rosmaric acid is the core precursor of salvianolic acid B. Most of the structural genes of phenolic acids biosynthesis pathway in Salvia miltiorrhiza have been cloned and studied, but study on the relevance of SmRAS to phenolic acids biosynthesis is still less. In order to explore vital function of rosmarinic acid synthase gene in biosynthesis of the water-soluble phenolic acids of S.miltiorrhiza, we designed an artificial miRNA(amiRNA) and subsequently generated an amiRNA overexpression plant construct, which was transformed into Salvia miltiorrhiza mediated by agrobacteria, aiming to silencing SmRAS gene. This research lay the foundation for using genetic engineering to improve the quality of Salvia miltiorrhiza.The main results were as follows:1. The nonconservative region of the SmRAS was selected as potential targetting site, an amiRNA/amiRNA* was designed by the WMD3 (Web microRNA designer) online to specifically slience SmRAS. Then amiRNA/amiRNA* was substituted for the miRNA319/miRNA319* duplex of Arabidopsis thaliana miR319a precursor using overlapping PCR to construct the amiRNA formation.2. Extract RNA of roots, stems and leaves of wild Salvia miltiorrhiza, using realtime quantitative PCR technology to analyze the expression quantity of SmRAS of Salvia miltiorrhiza roots, stems and leaves tissues. The result showed that SmRAS express to a different extent in different tissues of S. miltiorrhiza, where is highly in roots, moderate in stem, and the lowest in leaves.3. The amiRNA/amiRNA* precursor was transferred into wild Salvia miltiorrhiza by agrobacturium-mediated leaf disk transformation method, based on result of PCR validation for regenerative plants, we obtained 9 transgenic positive lines of amiRNA-SmRAS. Using semi-quantitative PCR, PCR-Southern blotting and realtime quantitative PCR, we analyzed the expression level of both SmRAS and amiRNA in positive lines. It was showed that the amiRNA was highly expressed in corresponding positive lines, meanwhile SmRAS expression level was generally obvious lower in positive lines compared to control, indicating that the designed amiRNA/amiRNA* precursor was successfully processed into mature amiRNA in transformed lines and subsequently lead to target gene silencing.4. Analyze the content of total phenolic in transformed lines and control showed that the total phenolic level in three transformed lines was obvious lower than control. The data in more details as follows:compared with control,3# decreased by 45.34%,6# decreased by 76.22% and 14# decreased by 28.48%.5. Analyze HPLC results showed that the content of both rosmarinic acid and salvianolic acid B were all reduced in 60d plants transformed lines. Out of these transformed lines,14# decreased most, respectively decreased by 49.80% and 32.95% compared to control.6. Compared with control, most of the transformed lines showed abnormal Phenotypes, such as delayed root formation, single and small taproots, more fibrous roots and so on. Besides, the roots looks like pink and the whole plant weaker than control.Conclusion:we generated an amiRNA construct to specifically silence the SmRAS gene of S.miltiorrhiza and obtained transgenic plants in this study. Results showed that the SmRAS gene played an important role in the synthesis of phenolic acids. This result provide new ideas for using genetic engineering to improve the content of secondary metabolites of S. miltiorrhiza.