Regulation Of Phenolic Acids And Tanshinones Biosynthesis By BHLH And WD40 Transcription Factors In Salvia Miltiorrihiza Hairy Roots

Salvia miltiorrhiza Bunge is one of the traditional Chinese herb medicines, which is famous for the prevention and treatment of cardiovascular and cerebrovascular diseases. It contains two major groups of bioactive ingredients, namely phenolic acids and tanshinones. Because of its widely needed, the quality of S. miltiorrhiza is drawing more attention than ever. About how to improve its quality have been an hot research area. In this study, we used S. miltiorrhiza hairy roots to study the regulatory mechanism of phenolic acids and tanshinones biosynthesis.1. S. miltiorrhiza hairy roots were treated with 15μmol·L-1 Ag+ and 100μmol·L-1 Me JA, respectively. Elicitation was performed on the 18 th day after inoculation. The hairy roots were harvested from the culture medium on days 1, 2, 3, 6 and 9 post-treatment. The content of active compounds in S. miltiorrhiza were studied by HPLC. And expressions of key genes in phenolic acids and tanshinones biosynthesis pathways were determined with RT-q PCR. The results showed that content of phenolic acids and tanshinones were both changed after treated with Ag+ or Me JA. Rosmarinic acid(RA), caffeic acid and ferulic acid were all induced by Ag+. However, contents of danshensu(DSU), cinnamic acid and salvianolic acid B(LAB) were significantly inhibited by Ag+. And accumulation of the total phenolic acids was almost not affected by Ag+ treatment. The four tanshinone components were induced by Ag+, and the total tanshinones accumulation was significantly induced by Ag+. It was indicate that tanshinones accumulation were more sensitive to Ag+ than phenolic acids. After treated with Me JA, all kinds of phenolic acids accumulation were dramticaly increased, especially the content of RA and LAB, and so as the total phenolic acids. However, tanshinoneⅠ(TⅠ) was almost not affected by Me JA treatment. And the accumulation of tanshinoneⅡA(TⅡA) was inhibited. But the content of cryptotanshinone(CT) and dihydrotanshinoneⅠ(DTⅠ) were induced by Me JA. As a whole, the affect of Me JA on total tanshinones accumulation was not obvious. Both Ag+ and Me JA can effect on the whole key genes of phenolic acids and tanshinones biosynthesis pathways. It was indicated that the effect of Ag+ and Me JA on secondary components was via affect on genes of the whole pathways. For tanshinones biosynthesis pathways, the MEP pathway was more sensitive than MVA pathway to the elicitors.2. Four b HLH and four WD40 transcription factor genes were isolated by the homology-based cloning and screening libraries, which were termed as Smb HLH7、Smb HLH8、Smb HLH9、Smb HLH10、Sm WD40-2、Sm WD40-5、Sm WD40-6 and Sm WD40-8. The eight genes harboured open reading frames(ORFs) with length of 2392bp、675bp、912bp、972bp、1740bp、1026bp、3396bp and 1338 bp, and coded 636、224、303、323、579、341、114 and 445 amino acids, respectively. All the four b HLH protein contain HLH domain, which belong to b HLH transcription family. And all the four WD40 amino acids sequence contain WD-repeat domain, which was indicate that the four WD40 protein belong to the WD40 transcription factor family.3. Overexpression vectors of the eight target genes were constructed and introduced into S. miltiorrhiza to get hairy roots with the Agrobacterium rhizogenes(ATCC15834) mediated genetic transformation system. We successfully constructed four b HLH-B34 and four WD40-B34 over-expression vectors, and transfered to Agrobacterium rhizogenes, respectively. And we used PCR to identificate the transgenic lines. Results showed that we have got four b HLH-B34 and four WD40-B34 over-expression S. miltiorrhiza hairy roots. The study was offered material for studying of the effect of transcription factors on active components biosynthesis in S. miltiorrhiza.