Isolation And Function Analysis Of BHIH Translation Factor In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is a kind of traditional Chinese medicine, which plays a crucial role in curing cerebrovascular diseases, irregular period, blood circulation and anti-cancer. There are mainly two types of bioactive components in S. miltiorrhiza, the hydrosoluble phenolic acids and liposoluble tanshinone which belong to diterpene quinines. Currently, because of its potential applications, S. miltiorrhiza metabolic engineering has become a hot spot.Jasmonic acid(JA) signaling pathway has been studied thoroughly in Arabidopsis thaliana. MYC2 transcription factor is a key component in JA signaling pathway, which plays an important role in the positive regulator of flavonoids(such as anthocyanins).Based on this, At MYC2 gene was isolated from A. thaliana firstly, and was introduced into vector p CAMBIA2300+ to construct the over-expression vector.The engineering strain C58C1 harboured the gene was transformed to S.miltiorrhiza to generate hairy roots. We find that At MYC2 can not only improve the tanshinones content, but also improve the Salvanic acid content(the tanshinones content of transgenic hairy At MYC2-3 was 16mg/g, which was 4.4 times higher than that in the control; while Salvanic acid content was 95mg/g, which was 3.6 times of the control group). The key enzymes of tanshinones and Salvanic acid synthesis pathway were also determined. We found that the expression level of these key enzymes were upregulated because of At MYC2 overexpression.Then Sm MYC2-like/Sm JAM3 were isolated from S. miltiorrhiza based on BLAST analysis, which encoding 604 amino acids and 491 amino acids respectively. Sm MYC2-like/ Sm JAM3 were introduced into vector p CAMBIA2300+ to construct the overexpression vector and p HB to construct knock-down vector. The engineering strain C58C1 harboured the gene was transformed to S.miltiorrhiza to generate hairy roots. Characterization and function were also investigated in this paper. Results of this stage are as follows:(1) Bioinformatics analysis: Sm MYC2-like/Sm JAM3 contain the b HLH domain, JID domain, NLS domains, and have close genetic relationship with At MYC2, At JAM3 respectively. Tobacco instantaneous transformation showed that Sm MYC2-like/Sm JAM3 expressed in the nucleus preferentially.(2) Tissues expression profile analysis: Sm MYC2-like expressed in taproot, fibrouroots, stems, old leaves, young leaves and petiole. But Sm MYC2-like had the highest expression level in the stems, followed by leaves, and had a lower expression level in the taproot, fibrous roots, old leaves. Sm JAM3 expressed in various tissues, but had a little difference of expression level among different tissues.(3) Induction analysis: Sm MYC2-like was induced by many elicitors such as MJ, YE, ABA, GA3, SA, ET, etc. just like At MYC2, and the effect is significant, indicating that Sm MYC2-like is likely to participate in a variety of hormonal signals pathway. Sm JAM3 was not induced by MJ, YE, ABA; SA had a more significant effect on the expression of Sm JAM3 while GA3 and ET had a weak effect.(4) Transgenic hairy root: The plant expression vectors of p CAMBIA2300+-Sm MYC2-like/Sm JAM3, p CAMBIA2300+-Sm MYC2-like-SRDX, and p HB-Sm JAM3-RNAi were successfully constructed. The plasmids of the vectors were introduced into A.tumefaciens strain C58C1. Finally, 14 Sm MYC2-like overexpression lines were attained while 16 knock-down lines were obtained. 11 Sm JAM3 overexpression lines were attained while 13 knock-down lines were obtained.(5) The expression profile of key enzymes: the expression level of the key enzymes of tanshinones and Salvanic acid synthesis pathway were also determined. Sm MYC2- like only had an effect on CYP76AH1 and GGPPS which were the key enzymes of tanshinones synthesis pathway, and up-regulated GGPPS, down-regulated CYP76AH1; while Sm MYC2-like only upregulate RAS in Salvanic acid synthesis pathway. Sm JAM3 did not have any effect on tanshinones synthesis pathway key enzymes, but down-regulated RAS expression.(6) Tanshinones content and salvanic acid content analysis:The average content of tanshinones and salvanic acid in S.miltiorrhiza hairy root lines was determined by HPLC. The average content of tanshinones in Sm MYC2-like and Sm JAM3 transgenic hair roots was higher than that in control lines, while salvanic acid content showed the same level when compared to the control.(7) Yeast two hybrid experiments: The results showed that Sm MYC2-like/ Sm JAM3 protein can interacte with Sm JAZ3/Sm JAZ9 protein respectively, indicating that the two genes involved in JA signal pathway.(8) Function validation in A. thaliana: In order to study the impact of the gene in the model plant A. thaliana, through genetic transformation in A. thaliana via agrobacterium GV3101, we obtained the transformed plants of the Sm JAM3. We found that Sm JAM3 could inhibit At MYC2 / At MYC3 / At MYC4 expression and promote At JAM1 expression, which caused the down regulation of At ERF1, At MYB75, At PDF1.2, At VSP2 expression.In conclusion, we cloned two S. miltiorrhiza MYCs genes, Sm MYC2-like/Sm JAM3, and investigated their founction, which would be helpful to uncover the metabolic mechanism of tanshinones and Salvanic acid synthetic and provide a theoretical basis to improve S. miltiorrhiza quality.