| Salvia miltiorrhiza Bunge is a well-known medicinal plant in the Labiatae family. The active constituents of S. miltiorrhiza can be divided into two groups: lipid-soluble tanshinones and water-soluble phenolic acids, its has been widely used for treatment of cardiovascular, cerebrovascular and heart disease. In recent years, accompanied by the growing demand of S.miltiorrhiza and a gradual reduction of its wild resources, improving the content of the active ingredients and cultivating new varieties with high quality have become the most urgent and key issues in the development of Salvia resources.The development of plant metabolic engineering through specific regulation of secondary metabolism was a promising alternative strategy for generating medicinal plants with enhanced health-promoting compounds. The strategy has two aspects: first, RNAi or overexpression of some key genes in the synthesis of secondary metabolites pathway, but plant metabolic pathway was a multi-step reaction with a variety of enzymes, so a single gene modification is difficult to work. The second strategy is based on the transcription factor, which can combine with cis-acting element to control the characteristics of the metabolic pathways. The third, through the secondary metabolites qualitative transport and accumulation of gene engineering technology research is an effective means to control secondary metabolites qualitative.There are many different kinds of salvia miltiorrhiza secondary metabolism substance, but its research in the accumulation of secondary metabolites qualitative transshipment and relative lag. ABC(ATP Binding Cassette)transporters is one of the super family in organism is widespread and nature across the membrane protein.ABC transporters in plant secondary metabolites qualitative transshipment and plays an important role in the process of accumulation, has become a key research direction of plant secondary metabolism engineering in recent years.Based on this, we cloned two ABC ransporters genes Sm ABCL1, Sm ABCL4. Sm ABCL1, Sm ABCL4 were introduced into vector p CAMBIA2300 to construct the over-expression and RNAi suppression vectors firstly.Theengineering strain C58C1 harboured the two genes were transformed to S. miltiorrhiza to generate hairy roots. In order to analyze the role of Sm ABCL1 and Sm ABCL4 in the tanshinones biosynthesis pathway, the expression levels of Sm ABCL1 and/or Sm ABCL4 were identified by QRT-PCR.HPLC was applied to analyze the tanshinone and phenolic acid in the transgenic hairy roots in order to clarify ABCs,effect on regulation of secondary metabolite.The results were as follow:1. The induction analysis of gene expression in S. miltiorrhiza: 100?M YE was added to one-month-old and normal growth hairy roots to testify the gene expression at 0Day, 2Day, 4Day, 6Day and 8Day. It showed that Sm ABCL1 initially responded to YE at 2Day and increased to the peak at 4Day after elicition; Sm ABCL4 didn't responded to YE.2. The tissues expression of Sm ABCL1 and Sm ABCL4 in S. miltiorrhiza: We compared and analyzed the relative expression level of Sm ABCL1 and Sm ABCL4 in tissues of S. miltiorrhiza such as leaf, stem, taproot, fibril, flower, petiole and seed. The results were as follow: Sm ABCL1 expressed more in root,leaf, petiole, taproot, fibril, stem hardly detected in seed, flower and seed. However, Sm ABCL4 can be detected in all the tissues, and it expressed the most in the seed less in petiole and fibril;3. Subcellular localization: The full length of Sm ABCL1 and Sm ABCL4 ORF were constructed into the expression vector p MON530-GFP. Tobacco instantaneous transformation showed that Sm ABCL1 expressed in the cytomembrane.4. Construction of plant expression vectors and preparation of engineering Agrobacterium tumefaciens: The plant expression vectors of p CAMBIA2300+-Sm ABCL1/Sm ABCL4 and p CAMBIA2300+-RNAi-Sm ABCL1/RNAi-Sm ABCL4 were successfully constructed. The plasmids of the vectors were introduced into A. tumefaciens strain C58C1;5. Plant transformation and Gene integration: The vectors were introduced into aseptic leaves of S. miltiorrhiza with A. rhizogenes C58C1 infection. 38, 43 and 36, 36 hairy root lines of Sm ABCL1, Sm ABCL4 and RNAi-Sm ABCL1, RNAi-Sm ANCL4 hairy root lines were attained, respectively. PCR analysis with primers located on 35 S promoter(35SF23)and genes' interior showed that 13, 16,14 and 16 transgenic lines were confirmed,respectively;6. Tanshinones content analysis:The average content of tanshinones in S. miltiorrhiza hairy root lines was determined by HPLC. The RNAi-Sm ABCL1 lines showed(7.54±3.19 mg/g DW) compared with the control(2.84±0.39mg/g DW)and the Over-expression Sm ABCL4 lines showed(1.73±0.45mg/g DW);7. Salvanic acidanalysis: HPLC was carried out to analysis the content of salvanic acidin S. miltiorrhiza hairy root lines. The Sm ABCL1 lines and Sm ABCL4 line showed almost the same production as in the control lines.We cloned two S. miltiorrhiza ABC genes:Sm ABCL1 and Sm ABCL4, we constructed the plant expression vectors and transformed them in S. miltiorrhiza hairy roots. HPLC analysis showed that Sm ABCL1 can down-regulate the tanshinone in S. miltiorrhiza hairy roots, which provided the scientific basis for improving tanshinones compounds contents and metabolizability. |
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