| Recently, application of fungal elicitor to stimulate the plant tissue and enhance the production of secondary metabolites is becoming a hot field. As a specific chemical signal, it can induce a variety of gene expression of plants rapidly, specifically and selectively. Especially it can activate the specific pathways of secondary metabolism, and accumulate the secondary metabolites. Endophytic fungi are widespread in the organization of health plant, and have developed a stable and symbiotic relationship with plant in the process of a long-term "co-evolution". A growing number of studies have shown that endophytic fungi could not only produce active molecules by themselves, but also adjust the production of plant secondary metabolites as biological inducers. But not much research on the mechanism of endophytic fungi promoting the synthesis and accumulation of plant secondary metabolites has been done.This research, based on years of screening active endophytic fungi strains from Sophora alopecuroides L. and inducing the callus and aseptic seedlings in the lab, established the system of suspension cell cultures of S. alopecuroides L., expecting to screen the endophytic fungal elicitors which can enhance the content of Kuinuolizidine alkaloids and then optimize the induction condition. On this basis, we preliminary discussed the effect of synthesis and accumulation of Quinolizidine alkaloids of endophytic fungi from S. alopecuroides L., which revealed the mechanism of synthesis and accumulation of Quinolizidine alkaloids by measuring the physiological defense reaction of S alopecuroides L. inducted by endophytic fungi. The main conclusions were as follows:1. The cotyledons and hypocotyls of S alopecuroides L. were taken as explants to study the optimum culture condition of callus induction and proliferation. In addition, the growth characteristics of suspension cell were investigated and the system of cell suspension culture was established preliminarily. The results showed that the most suitable materials for callus induction was cotyledon and hypocotyl explants, the optimal medium for callus induction was MS+2,4-D 0.5 mg/L+6-BA 1.0 mg/L+NAA 0.5 mg/L, the optimal formula for callus proliferation was MS+2,4-D 1.0 mg/L+6-BA 4.0 mg/L+NAA 1.0 mg/L. The callus inducted by hypocotyl was the most suitable material for suspension culture. It was effective to establish the suspension cell system when the initial inoculation was 10-20%, and sugar was 3%.2. Callus of S alopecuroides L. was taken as the experimental materials and run in HPLC for separation and determination of oxysophocarpine, oxymatrine, sophoridine, sophocarpine and matrine in callus of S alopecuroides L.. A chromatographic method was established for the separation and determination of five kinds of alkaloids from S alopecuroides L.. The results were summarized as follows:chromatographic conditions:Ultimate (?)AQ-C18(4.6 μm×250 mm×5 mm); mobile phase:0.01 mol/L phosphate buffer-Methanol(50:50 V/V); UV detection at 216 nm; column temperature:35℃; Flow rate:1.0 mL/min. There were five kinds of alkaloids in the callus of S alopecuroides L.. The contents of oxysophocarpine and oxymatrine are higher in subculture callus which are cultivating into stage 4. They are 1.205 6 mg/g and 0.116 5 mg/g. Sophoridine, The content of Sophocarpine and Sophocarpidine decreased as the time went by.3.8 strains of endophytic fungi were isolated and identified from S alopecuroides L. and their autoclaved mycelia and concentrated filtrates were prepared respectively to study the effect of different types, concentration and eliciting time of endophytic fungal elicitors on the growth of aseptic seedling and callus tissue of S. alopecuroides L., and the content of Quinolizidine alkaloids. Among the 8 strains of endophytic fungal elicitors, the effect of elicitation on the concentrated filtrate was stronger than those of the autoclaved mycelia. The autoclaved mycelia and concentrated filtrates of HMGKDFi could promote the growth of callus; the rate of growth was 1.82 and 1.42 times higher than that in CK, respectively. The concentrated filtrate of NDZKDF13 has a strong effect on the synthetic accumulation of Quinolizidine alkaloids of S. alopecuroides L.. The content of the total alkaloids was 0.5483 mg/g,23.8 times higher than that in CK; In a certain concentration range (0.01-1.0 mg/L), endophytic fungal elicitors of S. alopecuroides L.. could promote the synthetic accumulation of Quinolizidine alkaloids.4.4 strains of high-activity endophytic fungi of S. alopecuroides L. were preliminarily selected, and their concentrated filtrates were prepared as the fungal elicitors to investigate the effect on the physiological defense reaction and accumulation of Quinolizidine alkaloids. The results showed that:four strains of endophytic fungal elicitor of S alopecuroides L. could enhance the synthesis and accumulation of Quinolizidine alkaloids. Among them, endophytic fungi XKGKDF40 was most effective. The protein net growth of aseptic seeding was the highest, with a value of 142.35%,1.42 times higher than that in CK. The content of Quinolizidine alkaloids was 4.2101 mg/g,3.63 times higher than that in CK. The endophytic fungal elicitors could promote the activity of antioxidant enzymes, such as CAT, POD and SOD. Also they could promote the activity of PAL which was a key enzyme on the accumulation of alkaloids. |
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