| Salvia miltiorrhiza Bunge is a traditional Chinese medicine material, the hydrosoluble phenolic acids and liposoluble tanshinone which belong to diterpene quinines are two main types of bioactive components in S. miltiorrhiza. Tanshinones have many biological activity such as antioxidation, anticancer, anti-inflammatory, antibacterial activity and so on. At present, the hydrosoluble phenolic acids is one of the strongest antioxidant effect of natural products, which have a good effect on the treatment of many disease of heart head blood-vessel. However, the scarcity of the resources of wild Salvia miltiorrhiza, its quality degrades seriously, wild Salvia miltiorrhiza required long growth period, and the pharmaceutically active ingredient content is very low, so far can not meet the demand of the market. Metabolic engineering method was one of the practical way to improve the contents of tanshinones and phenolic acids, and the study about metabolic regulation of become the focus of Salvia miltiorrhiza study. Therefore, this study will provide some of the promise and foundation to carry out metabolic engineering of Salvia miltiorrhiza.Previous study showed that, plant specific AP2 / ERF transcription factors involved in regulation of many secondary metabolites and have an important role in many Stress and hormonal responses. The Sm ERF2 and Sm O3L3 transcriptional regulator were isolated from Salvia by in silico cloning method using the conserved domain amino acid sequenee of Cr ORCA3 as probe. Through studies including gene cloning, evolution study, expression analysis, promoter analysis, functional study of transgenic plants and gray mildew resistance of transgenic plants, the main results were achieved aslisted below:1. Sm ERF2 and Sm O3L3 ORF sequences were got by 3'RACE, they all have one AP2 domain and belong to ERF subfamily by bioinformatics alalysis. The structure analysis indicated that Sm ERF2 protein has a close affinity to At ERF2 and Aa ERF1(Aa ERF1 was reported to have a vital influence on Artemisinin synthesis) which means that Sm ERF2 may involved in the biosynthesis of Secondary metabolites of Salvia miltiorrhiza. Sm O3L3 protein has a close affinity to At RAP2.6L(ERF113).2. Tissue expression profile: compared the Sm ERF2 and Sm O3L3 expression profile among tissus of the wild Salvia plants, which have relatively highest expression at the leaves, stems, while have little expression in the petioles. But the expression differences between the organizations Sm ERF2 significantly smaller than Sm O3L3. Sm ERF2 and Sm O3L3 was significantly induced by methyl jasmonate(Me JA) and YE.But Sm ERF2 sensitive and can induced to higher levels, and Sm O3L3 response to these inducer slower.3. From the results of transformed instantaneously in tobacco, it is found that GFP:: ERF2 and GFP :: O3L3 c DNA fusion protein was located in the the nucleus.4. Analyzing the expression of some key enzyme genes identified in the PCRpositive transgenic hairy roots in the biosynthetic pathway showed that:in ERF2 overexpress hair root lines, Sm DXR, Sm DXS2, Sm HMGS, Sm CPS, Sm KSL related to tanshinones have relatively hight expression, But Sm HPPD have relatively lower expression. On the contrary Sm DXR, Sm DXS2, Gene expression levels in RNAi lines were on the contrary.5. In all transgenic roots, the overexpression Sm ERF2 lines(O) showed a higher tanshinones content than that of control, the average content was up to 6.488mg/g(dry weight) and was 1.58 times compare to the control(the content for the highest line could up to 9.2 mg/g and was 2.21 time to the control). This was consistent with the phenomenon that Sm ERF2 was induced significantly by YE(YE was known an effective elicitor to enhance tanshinones content). However, Sm O3L3 lines(O) have the higher salvianolic acid content with the control, the average content was up to 49.00mg/g(dry weight) and was 1.43 times compare to the control.6. The results of yeast one-hybrid showed that Sm ERF2 and Sm O3L3 could only bind with GCC-BOX instead of CBF and RAV eliments. It could preliminary got that Sm ERF2 and Sm O3L3 could just bind with A/G/CGCCGCC but not TGCCGCC.7. In addition, we also transformed Sm ERF2 and Sm O3L3 into Arabidopsis thaliana to research their possible function response to Pathogens and a serious of adversities.In summary, we cloned two ERF transcription factors, they all induced by YE and MJ, Sm ERF2 may have a significant positive effect on tanshinones content and Sm O3L3 was possible to improve salvianolic acid content. |
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