| The growth and accumulation of secondary metabolites of the medicinal plants are influenced by many factors, such as the external environmentl abiotic influence factor (temperature, illumination, humidity and et al.), and also effected by biotic impact factors, such as allelopathy, herbivorres, soil microorganism and plants endophytic fungi. Endophytic fungi are defined as the fungus that spend part or whole of their life-cycle colonizing inside the healthy tissues of roots, stems or leaves of the host plants, typically not causing obvious harms or apparent symptoms of disease to host plant. An increasing number of studies have indicated that endophytic fungi have significant effects on the growth, secondary metabolism and stress resistance to their host plants, which influenced their host mainly in the following aspects:First, promoting the biomass accumulation of host plant; Second, enhancing the biotic stress (pathogenic bacteria, animals and nematodes, etc.) and abiotic stress (high temperature, low temperature, drought, high salt and heavy metal) resistance of their host; Third, promoting the accumulation of bioactive metabolites of their host. At present, the mechanism of the effect of endophytic fungi on the host plants has not been fully elucidated, which limits the application of endophytic fungi in the cultivation and quality improvement of medicinal plants. As a traditional Chinese medicinal plant, Salvia miltiorrhiza possesses significant pharmacological activity in many aspects, mainly used to treat cardiovascular and cerebrovascular diseases, hypertension, atherosclerosis and cancer. The roots of S. miltiorrhiza contains two kinds of bioactive compounds:tanshinones and salvianolic acids. The former type mainly includes isotanshinone I, cryptotanshinone, tanshinone I and tanshinone Ⅱ A, while the latter type mainly includs salvianolic acid A, salvianolic acid B, salvianolic acid C and rosmarinic acid. The above two kinds of active components are the main material basis responsible for the bioactivities of S. miltiorrhiza. At present, the wild resources of S. miltiorrhiza is short due to excessive exploit, and the unstable genetic characteristics of the cultivated varieties make it easy to degenerate, resulting in uneven quality of the medicinal material. Cell and tissue culture technologies provide favorable conditions for investigating the secondary metabolism of S. miltiorrhiza. Especially, S. miltiorrhiza hairy root gets the advantages of genetic stability, rapid growth, plant hormone-autotrophic, which thus has become an ideal biological material to study the secondary metabolism of S. miltiorrhiza.Therefore, our study selected the endophytic fungi of S. miltiorrhiza as the research object and S. miltiorrhiza hairy roots as the experimental material, which was conducted to screen the target strains that could produce the same bioactive compounds as S. miltiorrhiza and promote the accumulation of secondary metabolites. HPLC and UHPLC-HRMS technologies are used to screen the host metabolite-producing target strains, whereas molecular identification is used to characterize and identify the target strains. HPLC has been used to analyze the secondary metabolites of S. miltiorrhiza hairy root treated with endophytic fungi. The infection of fungal endophyte in the hairy root tissue is examined by immunofluorescence staining. Finally, RT-PCR is used to analyze the expression of key enzyme genes involved in the metabolic pathway of S. miltiorrhiza, and explore the potential mechanism about the effect of endophytic fungi on S. miltiorrhiza hairy root metabolism, preliminarily providing experimental basis to reveal the mechanism of endophytic fungi influence on the quality of medicinal plants. The main results of our study are listed as follows.1. Isolation and purification of endophytic fungi1.1. The endophytic fungi were isolated from S. miltiorrhiza tissues (root, stem and leaf) collected from Shangluo, Shaanxi province. Through the traditional isolation and purification methods,123 strains of endophytic fungi were isolated from the tissues of S. miltiorrhiza. According to the morphological characteristics and color, the endophytic fungi were sorted into 80 morphotypes.2. Study on the target strains that can produce salvianolic acids2.1. A target strain that can produce salvianolic acid C has been screened and identified. By small amounts of liquid fermentation of endophytic fungi and methanol extraction, HPLC and UHPLC-HRMS was used to analyze the metabolites yielded by the endophytes, which resulted in the discovery that a strain D14 can produce salvianolic acid C. D14 was further identified as Phoma glomerata by molecular identification based on its 5.8S gene and ITS sequence. The preservation number of the endophytic fungus is CGMCC No.11305. So our study may provide a candidate engineering strain for salvianolic acid C production by microbial fermentation.2.2. The results of the co-culture of endophytic fungus D14 mycelium with hairy roots showed that D14 can not be co-cultured with S. miltiorrhiza hairy root for a long time. So we extracted D14 mycelium with water to prepare elicitor solution, which was used to be co-cultured with the hairy roots. The results show that D14 elicitor could not promote the accumulation of salvianolic acids components neither.2.3. Endophytic fungus D14 was fermented with PDB medium. Then the fermentation broth was extracted with methanol for further chemical composition investigation. As a result, a total of 3 compounds were isolated and determined, as glomeratanin A (PG-1, new), ergosterol (PG-2) and ergosterol peroxide (PG-3).3. Study on the target strains that can promote the accumulation of tanshinones3.1. S. miltiorrhiza hairy roots were cultured with endophytic fungi, and HPLC was used to analyze the content changes of secondary metabolites in hairy roots. We obtained a target strain D38 that can significantly promote the accumulation of tanshinones in S. miltiorrhiza hairy toors. The endophytic fungus D38 was identified as Chaetomium globosum by the molecular identification.3.2. The hairy roots were co-cultured with D38 mycelium and D38 elicitor, respectively. D38 mycelium could be co-cultured with hairy roots for a long time, and could significantly promote the accumulation of isotanshinone I and cryptotanshinone. After 18 days co-culture, the content of isotanshinone I increased 8 times, while that of cryptotanshinone increased 14.9 times, compared to the control group. Further,1/2B5 medium was used to prepare the low, medium and high concentrations of D38 elicitor solution, which were subsequently co-cultured with S. miltiorrhiza hairy for 18 days. The result showed that D38 elicitor could also promote the accumulation of tanshinones significantly, with the contents of isotanshinone I increased 21 times, cryptotanshinone increased 19.8 times, tanshinone I increased 1.2 times and tanshinone IIA increased 1 times, compared to control group, respectively. The results showed that both D38 mycelium and D38 elicitor could significantly promote the accumulation of tanshinones in S. miltiorrhiza hairy root and D38 elicitor is better.3.3. Studying the infection of D38 in S. miltiorrhiza hairy root tissues by immunofluorescence staining after being co-cultured with hairy root.The result indicated that most of the D38 mycelium colonied in the extracellular space, a small part was inside the cell. So we predict that D38 breeds in the extracellular space and gradually transfer to the inside of the cell. So it provided a preliminary experimental basis for studying the infection pathway of endophytic fungi in host plant.3.4. We study the expression of key enzymes in the tanshinones metabolic pathway of S. miltiorrhiza effected by D38 elicitor. Five key enzymes, including HMGR, DXR, GGPPS, CPS and KSL in metabolic pathway, were selected and their enzymes genes expression were determined by using real-time quantitative polymerase chain reaction (RT-PCR). The results indicated that D38 elicitor can significantly improve the expression levels of HMGR, DXR, GGPPS, CPS and KSL in tanshinones biosynthetic pathway. |
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