Effects Of Endophytic Fungus On The Growth And Secondary Metabolism Of Salvia Miltiorrhiza Hairy Roots And Its Molecular Mechanism

The environment of medicinal plant has played a decisive role on the quality of traditionalChinese medicine. However, in the past the abiotic factors in the environment such as light,temperature and humidity have been more concerned and the biotic factors such asmicroorganism have been ignored. Now more and more traditional Chinese medicineresearchers have realized that the biotic factors have very important effects on the qualityof traditional Chinese medicine. Endophytic fungus has been considered as one of thebiotic factors which have significant effects on the growth of medicinal plants and thebiosynthesis of bioactive metabolites in plants. At present, a thorough, in-depth andsystematic research on the effects of endophytic fungi on medicinal plant and the actionmechanism is rarely seen. Therefore, to a certain extent, the understanding of the effects ofendophytes on the herbal medicine quality and their application in the cultivation ofmedicinal plants have been restricted.In this study, the Salvia miltiorrhiza hairy roots are considered as a research platform toinvestigate the effects of the target endophytic fungus, which was isolated from S.miltiorrhiza and produced the same active constitutents as the host plant, on the rootgrowth and biosynthesis of active ingredients in the roots. Modern phytochemical separatemeans have been used to study the material basis of the endophytic fungus influencing onthe active ingredient biosynthesis in S. miltiorrhiza hairy roots and find out the activecomponents in the endophytic fungus. Technologies of metabolomics have been used tostudy the influences of the active components from the endophytic fungus on the metabolicprofiles of S. miltiorrhiza hairy roots and explore the regular patterns about theaccumulation of the metabolites in the roots. Means of transcriptomics have been appliedto study the influences of the active components from the endophytic fungus on thetranscriptional level of S. miltiorrhiza hairy roots and analyze changes in gene expressionprofiles. Proteomic techniques have been used to investigate the influences of the activecomponents from the endophytic fungus on the protein level of S. miltiorrhiza hairy rootsand find out the differentially expressed proteins. It is hopeful to elucidate the molecularmechanisms of the endophytic fungus influence on S. miltiorrhiza hairy roots and provideexperimental evidences and scientific basis for revealing the mechanisms of endophyticfungi influence on the quality of traditional Chinese medicine. The main results are asfollow:1. A total of103strains of endophytic fungi, which were mergered into56morphotypes according to the morphological characteristics, were isolated from the cultivated S.miltiorrhiza from the city of Shangluo in Shanxi province and the city of Bozhou in Anhuiprovince and the wild S. miltiorrhiza from the city of Shangluo as well. The means ofHPLC, LC-MSnand UHPLC-HRMS were used to analyze the secondary metabolites ofthese endophytic fungi and one isolate D16, which was identified as Trichodermaatroviride by ITS analysis (ITS1and ITS2regions and the intervening5.8S rDNA region),was found out to produce tanshinoneⅠand tanshinoneⅡA. This finding has provided atarget strain to research the effects of endophytic fungi on S. miltiorrhiza hairy roots and analternative engineered strain to obtain tanshinones through microbial fermentation.2. The mycelium of D16was added to the culture medium of S. miltiorrhiza hairy rootsand the effects of the mycelium on the root growth and the biosynthesis of secondarymetabolites in the hairy roots were investigated. The results showed that in the co-cultured5days the mycelium of D16had no significant effects on the root growth, and had nosignificant impacts on the contents of phenolic acids in the early days yet, but stronglyinhibited the contents of phenolic acids on day5. Meanwhile, the contents of tanshinoneswere significantly increased.3. In order to remove the virulence of the mycelium and to study the effects of D16on S.miltiorrhiza hairy roots in a long period, the water-soluble extract of D16myceliumconsidered as an elicitor was added to the culture medium of S. miltiorrhiza hairy roots andthe influences of the elicitor on the root growth and the biosynthesis of secondarymetabolites in the hairy roots were researched in18days. The results showed that theelicitor from D16signifisantly enhanced the biomass of S. miltiorrhiza hairy roots andpromoted the biosynthesis of tanshinones notably. Meanwhile, the biosynthesis of phenolicacids was inhibited slightly. This study indicated that D16had notable influences on thegrowth and active component accumulation of S. miltiorrhiza hairy roots.4. The preliminary study about the material basis of the endophytic fungus promotingthe biosynthesis of tanshinones in S. miltiorrhiza hairy roots was carried out through themodern chemical separate means. It was foud that protein polysaccharide fraction (PSF)was the main active fraction to promote tanshinone biosynthesis. Another5endophyticfungi from S. miltiorrhiza were selected to study the specificity about the tanshinonebiosynthetic promotion by D16. The results indicated that all the PSFs from the6strainshad the effects of promoting tanshinone biosynthesis, but the action intensity of PSF fromD16was much stronger than others’. 5. The changes of the S. miltiorrhiza hairy root metabolic profiles induced by PSF fromD16were investigated by metabolomics technology based on UHPLC-HRMS. The resultsof principal component analysis and cluster analysis suggested that the metabolic profilesof S. miltiorrhiza hairy roots were notably changed by PSF from D16. A total of33differential metabolites, which included10tanshinones,6phenolic acids,4amino acids,6organic acids and7another components, were identified through high-resolution molecularion peaks, fragment ion peaks, public databases and literatures. After quantitative analysis,it was found out that PSF from D16promoted the biosynthesis of tanshinones notably andinhibited the biosynthesis of phenolic acids slightly.6. A large scale functional genomic database of S. miltiorrhiza hairy roots wasestablished through high-throughput next-generation sequencing technology andbioinformatics analysis.121353unigenes with an average length of825bp were obtainedafter de novo assembly and48588unigenes were annotated by the public databases andinvolved in the main vital movements of plants. Through differential expression analysis ofthe S. miltiorrhiza hairy root transcriptomes with/without PSF treatment,2307unigeneswere found out to be differential expressions. With deep analysis of these differentialexpression unigenes, it could be presumed that PSF from D16induced S. miltiorrhiza hairyroots to produce strong defense responses firstly. And then through the differentialexpressions of the genes in the signal transduction pathways involved in calcium, oxidativestress, ethylene, jasmonic acid and salicylic acid, the genes such as transcription factorwere differentially expressed. After that, the expressions of genes involved in the processof secondary metabolites such as oxidation-reduction process, terpene biosynthesis andphenolic acid biosynthesis were changed. Finally, the metabolic profiles of S. miltiorrhizahairy roots were changed notably.7. The protein changes of S. miltiorrhiza hairy roots treated by PSF from D16werestudied by proteomics technique of iTRAQ. A total of1476proteins were identifiedaccording to the database of Arabidopsis thaliana, and89differential expression proteinswere picked out by quantitative analysis. With deep analysis of these differentialexpression proteins, it could be presumed that PSF from D16regulated the synthesis andmetabolism of saccharides and amino acids, the transcription of genes and the translationof protein through the signal transduction pathways involved in leucine-rich repeat proteins,calcium, peroxides, protein phosphorylation and jasmonic acid synthesis, and then resultedin expression changes of the proteins involved in secondary metabolism, and induced the metabolic profiles of S. miltiorrhiza hairy roots to change notably at last.