Transcription Factor Pap2 Danshen Phenolic Acids Biosynthesis

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 phenolic acids 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. For a long time, the phenolic acids from S. miltiorrhiza were considered the main components in water-decoction, which is the major form administered to patients in clinical medication of China. At present, many means have been employed for enhance the contents of phenolic acids based on hairy root cultures and changes in ploidy, but the result were not good. 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. Previous studies have showed that, MYB transcription factor involved in phenylpropanoid metabolism regulation widely, the molecular biology had been applied to regulate the expression of specific transcription factors to enhance the accumulation of specific compounds. Preliminary studies in our laboratory found that many genes which contain MYB binding sites were related to the synthesis of phenolic acids. In view of the above-mentioned facts, we speculate that MYB transcription factors may have an important regulatory role in the synthesis of phenolic acids. In order to provide some evidences of breeding fine strains for Salvia, so we choose the MYB90 family transcription factors PAP 2 in Arabidopsis to overexpress in S. miltiorrhiza, screening of some strains which the phenolic components were significantly improved.The main results were as follows:1. The 750 bp fragments, located in the 3'end of the coding region of PAP2genes, were chosen for overexpression investigation, which was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transformation, and 9 transgenic lines were obtained by PCR screening.2. Compared with the controls, analysis of transcription quantity by real-time PCR showed the high transcription and translation in three transgenic lines.3. Water extracts of three transgenic lines were chosen for HPLC analysis. The PAP 2 roots and leaves, compared with control plants, shows difference. The contents of danshensu, salvianolic acid B and rosmarinci acid in transgenic lines were significantly higher than those in control,while the content of caffeic acid was not predominant.4.PAP2 has an additional, previously unknown role as a transcriptional activator of phenolic acid biosynthesis in S. miltiorrhizaPAP2 activated a broader spectrum of genes, such as PAL, C4H,4CLand TAT in the phenylpropanoid pathway. It is effective in enriching the formation of phenolic acids in transgenic S. miltiorrhiza.Besides, PAP2 induced F3'Hand F3'5'Hhigh transcription and enhanced the metabolic flux in anthocyanin pathway.5. The total lignin, the Klason lignin and acid lignin in transgenic plants were detected. The results show that:Klason lignin and acid lignin were decreased in the transgenic lines, related to the decrease of CCR and COMT gene expression.6. Total phenolic, total flavonoid and anthocyanin contents were detected. The results showed that the contents of three ingredients increased a little.In conclusion,9 transgenic lines were obtained by PCR screening. Phenolic acids level increased diversely in leaves and roots. The total phenolic, flavonoid and anthocyanin increased slightly, while the content of lignin decreased but not significant. This research is of great significance for the further studies of the secondary metabolite regulation and molecular breeding of S. miltiorrhiza, and provides a promising strategy for genetic engineering of other medicinal plants.