| Tripterygium wilfordii Hook.f.(Celastraceae),woody climber,has important agriculcural activity and medical value.Extensive studies had been widely carried out since it can produce a series of biological active substances,including triptolide and alkaloids.Under the double pressure of the competition of the medical resources and the slow breeding of tripterygium plant itself,the resource problems of tripterygium alkaloids pesticide become more prominent.With the dramatically increasing demands for medicine and biorational pesticide,and the blind exploitation of wild resources,it's an effective way to produce useful metabolites through tissue culture to cope with the problem.This study begin with an endophyte(F4-20)isolated from tripterygium wilfordii root.Wilforgine is contained in secondary metabolites confirmed by HPLC and LC-MS/MS.And in order to improve the insecticidal activity and Tripterygium wilforgine content,as well as to prepare for the production of industrial fermentation,the fermentation conditions of endophyte culture were optimized.The main results and conclusions were given as follows:1.To finding endophyte's best growth conditions,we cultured solid medium at different temperatures,carbon,nitrogen,initial pH and auxin.The results showed that the optimal temperature was 20 ?,the best initial pH was 8.5 and the best auxin was NAA.Among different carbon sources in culture medium,when Glucose was the carbon source,mycelia growth was the fastest,coming to 4.47 cm after culturing 20 days.Among different nitrogen source mediums,when Bean Flour was the nitrogen source,mycelia growth was the fastest,achieving 5.54 cm after culturing 20 days.2.Using HPLC and LC-MS/S detection,the results show that the secondary metabolites of F4-20 contains wilforgine.Using E medium(ISP2),wilforgine was up to 44.2?g/g in F4-20 secondary metabolites.3.With the insecticidal activity and mycelium dry weight as indicators,we screened the basal medium of strain F4-20.The results showed that medium H was better than the others,from which mycelium extract exhibited the highest lethal rate of 5.67%on 3rd instar larva of mosquito and 58.34%on aphids.The mycelium dry weight was 8.89 mg/ml after 7d cultivation.on the basis of the best carbon source and nitrogen source,and with the mycelial dry weight as an indicator,F4-20 fermentation medium were optimized by using response surface methodology.The results showed that when glucose 35.7 g/L,peptone 26 g/L,MgSO4·HO 0.059 g/L,NaCl 2.38 g/L,KH2P04 1.19 g/L,CaC03 2.38 g/L,and(NH4)2SO42.97 g/L,it was most conducive to the growth for F4-20,and mycelium dry weight reached up to 13.05 mg/mL after 7d cultivation.On this basis,with mycelium dry weight as an indicator,by using response surface analysis on different initial pH,medium volumes,the inoculation interaction and the rotate speeds,the flask-shaking fermentation conditions of strain F4-20 were optimized.The study showed that when the initial pH 7.5,inoculation 9.04%,liquid volume 94.2mL/250mL and the rotate speed 140 r/min,the maximum dry weight of F4-20 was 15.93 mg/ml after 7d cultivation.4.Four plant stress molecules,MeJA,SA,H2O2 and GSH were applied separately into the invitro culture of F4-20 to examine their effects on the bacterial growth and production of wilforgine.Results showed that MeJA,SA,H2O2 could inhibit the growth in different degrees and significant increase in growth was observed soon after GSH treatment.The growth of F4-20 was significantly induced by GSH.The maximum of mycelia fresh weight of 4.72±0.40 g,1.39 fold of the control(3.40±0.07 g),was observed after 12h treatment of 3 mM GSH.The contents of wilforgine in the mycelia of F4-20 were reduced by the addition of MeJA and GSH,but induced by SA and H2O2 after 12h.After adding 100 uM SA,wilforgine was up to 45.91 ± 1.26 g,1.42 fold of the control(32.39±2.61 ?g/g).5.Endophytes F4-20 were fermented by 5 L,15 L and 70 L fermenters,the results show that different mixing speed,aeration and fermenters volume had a greater affect on the growth and metabolism tripterygium of endophytes F4-20.When the mixing speed 200 rpm/min,aeration rate 2.5 L/min by the 5 L fermentor,the maximum dry weight of F4-20 was 12.94 mg/ml after 10d cultivation,and it benefited the growth and metabolism.When mycelial dry sample concentration was 0.15 g/mL,the insecticidal activity of Macrosiphum roswomm Zhang was 56.61%.When the mixing speed 200 r/min,aeration rate 10 L/min by the fermentation medium and ISP2 in the 15 L fermentor,the maximum dry weight of F4-20 were 6.02 mg/mL and 2.02 mg/mL.When mycelial dry sample concentration was 0.15 g/mL,the insecticidal activity of Macrosiphum roswomm Zhang were 47.09%and 52.87%.When the mixing speed 150 r/min,aeration rate 40 L/min by the fermentation medium in the 70L fermentor,the maximum dry weight of F4-20 was 6.02 mg/mL and 2.43 mg/mL.When mycelial dry sample concentration was 0.15 g/mL,the insecticidal activity of Macrosiphum roswomm Zhang was 37.57%.6.The 16S rDNA complete sequences of F4-20 were amplified PCR.The homology comparison and clustering analysis of the 16S rDNA sequences of the endophyte were conducted by comparing with the known strains in GenBank.In constructed phylogenetic tree,F4-20 formed a monophyletic clade with strains of streptomycete Blastomycetica strain NRRL B-5480 with sequence homology higher than 98%.Combined with the early identification results of physiological and biochemical characteristics,F4-20 o belongs to the Streptomyces genus of actinomycetes. |
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