Transcriptional Self-activation Of Salvia Miltiorrhiza Transcription Factor Gene SmPAP1 And Its Regulation Of Secondary Metabolic Pathways Of Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge (S. miltiorrhiza) which belonged to Labiatae family, is a very popular medicinal plant in China. Its air-dried roots are used for the prevention and treatment of various diseases. Phenolic acids as the main medicinal ingredients of S. miltiorrhiza get more and more attention.Studies on SmPAP1 belonged to R2R3-MYB transcription factor showed that, SmPAP1 does serve as an important regulatory protein to regulate the metabolic pathway of phenolic acids in S. miltiorrhiza. Preliminary studies focused on transcriptional regulation on its downstream target genes. But, the transcriptional regulation mechanism of SmPAP1 itself in S. miltiorrhiza is still not clear. Analysis of the conserved DNA sequence elements of SmPAP1 promoter showed that, there are multiple potential MYB cis-acting elements in the promoter of SmPAP1. Therefore, we speculated that SmPAP1 may act as a positive regulatory factor that can bind to the promoter of its own to activate itself expression. Therefore, a reporter vector containing ?-glucuronidase (GUS) that was driven by SmPAP1 promoter was used in this study. GUS histochemical staining and GUS activity analysis were used to explore whether SmPAP1 can activate its own promoter expression or not. In order to analyse the molecular function and mechanism of SmPAP1 affects the synthesis of the effective constituents in S. miltiorrhiza, transcriptome analysis were taken between SmPAP1 over-expression lines and wild type.The main results of this study are as follows:1. Reporter(SmPAP1-Promoter::GUS) and effector (35S::SmPAP1) were co-transformed into leaves of N. benthamiana by transient tobacco expression system to explore whether overexpression SmPAP1 in tobacco cells could activate its own promoter or not. GUS histochemical staining and GUS activity analysis showed that overexpression of SmPAP1 can increase GUS gene expression and GUS protein activity. Compared to control, protein activity was increased about 5.15?15.50 fold. The results showed that SmPAP1 could activate its own promoter in tobacco cells.2. The SmPAP1-Promoter::GUS vector was transferred into S. miltiorrhiza using Agrobacterium-mediated transformation medthod. We obtained 9 overexpressed tansgenic lines,16 RNAi tansgenic lines and 7 wildtype tansgenic lines, according to the results of PCR screening. Expression of GUS in transgenic S. miltiorrhiza lines was detected by the Real-time quantitative PCR, the results showed that:the contents of GUS gene in all the overexpressed transgenic lines were higher than control. While the contents of GUS gene in all the RNAi transgenic lines were significantly reduced compared to control. GUS histochemical staining and GUS activity analysis of transgenic lines showed that:GUS activity of overexpressed transgenic lines were about 1.43-2.13 fold of control lines, respectively while GUS activity of RNAi transgenic lines were about 1.30%?2.99% of control lines.3. To further explor the molecular function and mechanism of SmPAP1 that affects the synthesis of the effective constituents in S. miltiorrhiza, transcriptome analyses were taken for comparing SmPAPl over-expression and wild-type. Compared to wild-type, 1,559 different expression genes (DEGs) were selected out. Among them,566 genes were up-regulated and 993 were down-regulated. Gene Ontology enrichment analysis showed that DEGs were involved in 69 GO term, which 25 in biological process,38 in molecular function and 6 in cellular component. DEGs were enriched in catalytic activity and response to stress. Pathway enrichment analysis showed DEGs were involved in 75 pathways. Among them,9 pathways were enriched, including phenylpropanoid biosynthesis, flavonoid biosynthesis, and diterpenoid biosynthesisand so on. Among them, biosynthesis of secondary metabolites has the largest number. SmPAPl activated PAL and TAT in the phenylpropanoid pathway and increased the formation of phenolic acids in S. miltiorrhiza. Besides, SmPAP1 reduced CPS transcription and affected lipid-soluble tanshinones in transgenic plants.This research made a preliminary exploration of the self-activating regulation of transcription factor SmPAP1. Results showed that there exists self-activating regulation of SmPAPl in S. miltiorrhiza. Besides, SmPAP1 may enrich the formation of phenolic acids by regulating PAL and TAT in the phenylpropanoid pathway in S. miltiorrhiza. This research is of great significance for the further studies of regulation mechanism and molecular function of SmPAP1.