| Panax ginseng C.A.Mey.,one of the medicinal plants of the genus Panax,belongs to Araliaceae.The medicinal parts of this plants are dry root and rhizome.Firstly recorded as the top grade in Shennong's Classic of Meteria Medica,P.ginseng is commonly used traditional Chinese medicine in our country.The medicine tastes sweet,slightly bitter and the effect is mild moderate.In lung,spleen,heart and kidney meridian,P.ginseng helps to replenish energy,tonify spleen and lung,generate saliva,stop cough,soothe nerves and benefit the kidney.It is mainly used in the treatment of Heart-Qi Deficiency,coronary artery disease and arrhythmia clinically,which is one of the most widely used and deeply sdudied Traditional Chinese medicine.It mainly distributed in eastern Liaoning province,eastern Jilin province and eastern Heilongjiang province in China.Modern research has shown that ginseng contains ginsenosides,amino acids,volatile oil components and etc.,and the recognized effective components are mainly ginsenosides,which can be divided into three types: one is oleanane triterpenoidal saponins like ginsenoside Ro;one is panaxadiol dammarane tetracyclic triterpenoids like ginsenoside Rh2,ginsenoside Rg3,ginsenoside Rb1,ginsenoside Rb and ginsenoside Rc;the other one is panaxatriol dammarane tetracyclic triterpenoids like ginsenoside Re,ginsenoside Rf and ginsenoside Rg1.P.ginseng hairy roots can be induced by using Agrobacterium rhizogenes infecting the cells of the host plants P.ginseng.Compared with the traditional cell culture technology,hairy root is more ideal in the production of secondary metabolites of plant tissue,because of its short growth cycle and stable genetic traits and hormone autotrophic etc,and also eliminates the dependence on exogenous plant hormone in traditional cultures.P.ginseng hairy roots is a hormone autotrophic plant tissue,and it is meaningful to change the second metabolites accumulations by regulation.Thus,for the production of secondary metabolites such as ginsenosides and ginseng polysaccharide with the platform of P.ginseng hairy roots opens up a new way.In this topic,the endophytic fungi of important medicinal plants in our country,Panax ginseng C.A.Mey.and Panax quinquefolius L.were set as the research object;the P.ginseng hairy roots were set as the research platform.The target endophytic fungi which can promote the growth of host plants and stimulate the effective components contents were screened out;the target endophytic fungi were identified through the morphological and molecular identification;the changes on the active ingredients of P.ginseng hairy roots wre determied by high performance liquid chromatography(HPLC)technology;the infection process of the endophytic fungi on the host plants were observed by immunofluorescence staining techniques;the changes of ginsenosides key enzyme gene in metabolic pathways were calculated through the RT-RCR technology.These reliminary studies were expected to discuss the mechanism of endophytic fungi on P.ginseng hairy root and provide experimental foundation and scientific basis for revealing how endophytic fungi affect Chinese medicinal plants and its mechanisms.The main results were as follows:1.83 endophytic fungi were isolated from 3 years cultivated P.ginseng,4 years cultivated P.ginseng and 4 years cultivated P.quinquefolius from Jilin tonghua by using traditional separation method,and these endophytes were mergered into 55 morphotypes according to the morphological characteristics;a strain of endophyte 3R-2 that can co-culture with P.ginseng hairy roots and enhance the biomass and contents of ginsenoside Rc,ginsenoside Rg2 and ginsenoside Rg3 of P.ginseng hairy roots was screened out by using HPLC,which was identified as Schizophyllum commune through the morphological and molecular identification.The screening of P.ginseng endophytes provides target strains for the study of the effects of endophytes on P.ginseng in the next step.2.The endophyte 3R-2 hyphae was added into of P.ginseng hairy root cultures by means of punching,the effects of endophyte 3R-2 hyphae on the growth and secondary metabolic biosynthesis of P.ginseng hairy roots were studied.Results show that the endophyte 3R-2 has no obvious effects on the biomass and contents of ginsenoside Rc,ginsenoside Rg2 and ginsenoside Rg3 of P.ginseng hairy roots within seven days,and it significantly promote the biomass and contents of ginsenoside Rc,ginsenoside Rg2 and ginsenoside Rg3 of P.ginseng hairy roots within 14-21 days;the content of ginsenoside Rc reached the highest in 14 days endophyte 3R-2 hyphae co-culture with P.ginseng hairy roots.3.In order to remove the virulence of the endophyte 3R-2 hyphae to examine the influence of 3R-2 on P.ginseng hairy roots,water-soluble extracts of endophte 3R-2 hyphae was added into P.ginseng hairy roots as an elicitor to study the influence of which on the growth and ginsenosides biosynthesis of secondary metabolism.The results show that the 3R-2 elicitor played a certain role in promoting P.ginseng hairy roots biomass,and it is clear that endophyte 3R-2 has the important influence on growth and the accumulation of active ingredients of P.ginseng hairy roots.4.The existence of endophytes and the morphology of endophytes were obviously observed in fresh roots,stems,leaves and fruits of Changbai mountain cultivated P.ginseng and P.ginseng hairy roots through P.inseng tissues embedding slicing,HE ammonium silver staining and immunofluorescence staining.The mycelium in stems and fruits of P.ginseng observed under the microscope is the most obvious;the infection situation of several strains important endophytic fungi in P.ginseng hairy root tissues was observed and the main infection sites were found in parenchyma cells around.The means of endophytic fungi infection on plant tissues may provide some experimental basis for the future research.5.The rt-PCR of four key enzyme genes HMGR,SS,SE and DS in the secondary metabolic pathways of ginsenosides was carried out,and the results showed that key enzyme genes SS of the 3R-2 mycelium group is significantly higher than the control group and reached the highest in 6 days,while the key enzyme genes HMGR,SE and DS of the 3R-2 elicitor groups reached its highest in 15 days and were 90 times,20 times and 120 times higher than control group,respectively.6.Our study made clear that 3R-2 hyphae influenced greatly on P.ginseng hairy roots and played a key role in ginseng saponins biosynthesis by using metabonomics methods;the P.ginseng hairy root methanol extracts of 3R-2 treatment group and the blank control group at different time points from positive and negative ion mode mass spectrometry were processed,and 2166 compounds were obtained from positive ion mode and 2189 compounds were obtained from negative ion mode.Heatmap showed that large amounts of compound significantly increased or declined after being treated;PCA analysis found that with the increase of processing time,the metabolism difference was more significant. |
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