| Medicinal Salvia miltiorrhiza ("Danshen" in Chinese) is a perennial plant which belongs to Labiatae family. As a traditional medicine, its roots have important biological activities, including antioxidant, antitumor, and antimicrobial properties."Danshen" is renowned for its curative effects on coronary heart diseases, particularly angina pectoris and myocardial infarction. In recent years, accompanied by the growing demand for Danshen and a gradual reduction of its wild resources, to improve the content of the active ingredients and cultivate new varieties with high quality have become the most urgent and key problems in the development of Salvia resources. The active pharmaceutical ingredients of S. miltiorrhiza are divided into two main groups:water-soluble phenolic acids, such as caffeic acid, danshensu (3,4-dihydroxyphenyllactic acid), rosmarinic acid, salvianolic acids and lithospermic acid, and lipid-soluble tanshinones such as tanshinone I, tanshinone IIA and cryptotanshinone. The phenolics now attract more attention because they are the main components of water decoction, which is the most common form of dosing administered to patients in Chinese clinics. Rosmarinic acid is thought to be the core structure of most hydrophilic compounds in S. miltiorrhiza, such as salvianolic and lithospermic acids. A proposed biosynthetic pathway in Coleus blumei suggests that rosmarinic acid is an ester of3,4-dihydroxyphenyllactic acid and caffeic acid. Those two compounds are synthesized via the tyrosine-derived pathway and the phenylpropanoid pathway, respectively. At present, genes encoding the enzymes in the rosmarinic acid biosynthesis pathway including phenylalanine ammonia-lyase (PAL), cinnamate4-hydroxylase (C4H),4-coumarate:coenzyme A ligase (4CL), tyrosine amino-transferase (TAT) and hydroxyphenylpyruvate reductase (HPPR) have been cloned in S. miltiorrhiza. But the regulation genes of these enzymes are still unclear. The reports for the contribution of these genes to rosmarinic acid accumulation are also lacking. In this study, to gain insight into the nature of rosmarinic acid biosynthesis, we further cloned the genes which encoded the enzymes and transcription factors in the rosmarinic acid pathway in S. miltiorrhiza. The relationship between the expression levels of these genes and the accumulations of rosmarinic acid and salvianolic acid B were also analyzed. Furthermore, RNAi was used to study the function of the enzyme genes in the biosynthesis of the active water-soluble phenolic acids. Moreover, the key genes for the synthesis of rosmarinic acid and its derivatives were screened, which is of great significance for the further studies of the regulation of the secondary metabolites and molecular breeding of S. miltiorrhiza. The main results and conclusions are as follows:1. The entire sequence named MYB4, which belonged to R2R3-MYB transcription factor gene family, was cloned in S. miltiorrhiza by PCR. The Genbank accession number was GU586494. MYB4in S. miltiorrhiza consisted of two exons and one intron, and contained an open reading frame (ORF) of693bp length that encoded a protein of230amino acids. The amino acid sequence contained two conserved MYB DNA-binding domains, as well as the conserved motif of MYB4transcription factor (LNLDL). Sequence analysis showed that it shared high identity with MYB4and MYB32in Arabidopsis thaliana, which were two negative regulatory factors for C4H. The expression pattern of MYB4gene was analyzed by real-time quantitative PCR. The results indicated that it expressed in all S. miltiorrhiza organs but most highly in leaves. Methyl jasmonate (MeJA) could inhibit the expression of MYB4, while light and abscisic acid (ABA) can induce its expression in S. miltiorrhiza. MYB4shared contrary expression pattern with C4H at different stages of these treatments, suggesting that the transcription factor encoded by MYB4may function as the repressor of C4H in S. miltiorrhiza.2. RAS-like gene and its5’flanking sequence were cloned in S. miltiorrhiza by degenerated PCR and DNA Walking method. The Genbank accession numbers were GU647199and GU647200respectively. RAS-like gene contained an ORF of1284bp length encoding a protein of427amino acids with typical characteristics (conserved HXXXD motif and DFGWG motif) of the BAHD acyltransferase superfamily. It shared high identity with the rosmarinic acid synthase (RAS) in C. blumei. RAS-like gene expressed in roots, stems and leaves of S. miltiorrhiza but most highly in stems. Totally,900bp5’flanking region of RAS-like gene was obtained and the putative cis-elements in this region including pathogen responsive element (Box Wl), element involved in the MeJA-responsiveness, and a variety of light responsive elements were predicated. Based on this, the expression pattern of RAS-like gene was analyzed by real-time quantitative PCR. The results showed that the expression of RAS-like gene could be induced by Psoudomonas lachrymans, MeJA, light and salicylic acid (SA). Suppressing of RAS-like gene in S. miltiorrhiza via RNAi can lead to the reduction of rosmarinic acid and salvianolic acid B. But the DPPH radical scavenging activity and the contents of total phenolics and total flavonoids did not change significantly. These results indicated that RAS-like gene may play a role in the biosynthesis of rosmarinic acid in S. miltiorrhiza as the rosmarinic acid synthase gene.3. The5’flanking sequence of C4H was cloned in S. miltiorrhiza by DNA Walking. The Genbank accession number was GQ896332. Based on this, the cis-acting elements in the promoter regions of the rosmarinic acid-related genes(PAL, C4H,4CL, TAT, HPPR and RAS-like gene) in S. miltiorrhiza were analyzed, compared and classified. The results showed that light responsive elements were the most abundant elements and existed in the promoter regions of all rosmarinic acid-related genes. MYB-binding site and elements involved in MeJA and ABA responsiveness also existed in the promoter regions of the majority genes. These results indicated that light, MeJA, ABA and MYB transcription factors can influence the accumulation of rosmarinic acid and its derivatives by regulating the enzyme genes in the rosmarinic acid pathway at the same time.4. Gene expression levels and the accumulation of rosmarinic acid under the treatment of light or MeJA were detected by real-time quantitative PCR and high performance liquid chromatography respectively. Furthermore, a "gene-to-metabolite" network was constructed according canonical correlation analysis. By analyzing the relation between gene expression and metabolite accumulation, we found that PALI, PAL2, C4H,4CL2and HPPR were the key genes for rosmarinic acid biosynthesis under light treatment; PALI, C4H and HPPR were the key genes for the biosynthesis of salvianolic acid B under light treatment.5. Expression of PAL, C4H,4CL, TAT and HPPR were silenced in S. miltiorrhiza by RNAi to discuss the function of these genes in the process of antioxidant and the accumulation of water-soluble phenolic acids. The results showed that suppressing the expression of each gene can reduce the DPPH radical scavenging activity and the contents of total phenolics and total flavonoids, indicating that all the above genes played important roles in the metabolic course of the active ingredients in S. miltiorrhiza. The contents of rosmarinic acid and salvianolic acid B in different RNAi lines were also detected. Among them, the influence of TAT was the most significant, followed by PAL. After comparing between the RNAi lines for different genes, we concluded that PALI,4CL2and the RAS-like gene cloned here were the key genes for rosmarinic acid biosynthesis under non-induced condition. While TAT was the key gene for the biosynthesis of both rosmarinic acid and salvianolic acid B under non-induced condition. |
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