Screening And Optimization Of Alkaloid-producing Endophytic Fungi Of Sophora Alopecuroides L. For Quinolizidine Alkaloids

Sophora alopecuroides L.is a perennial herb in the genus Sophora of the family Leguminosae,mainly distributed in the desert and semi-desert areas of northwest China,and is a pioneer plant in natural pastures and also an important native medicinal herb of Ningxia.Its important active ingredients are Quinolizidine alkaloids and flavonoids,which have various effects such as clearing heat and detoxifying,dispelling wind and drying dampness,relieving pain and killing insects as well as enhancing immunity.The way to obtain the active ingredients of Sophora alopecuroides L in production mainly relies on the extraction of Sophora alopecuroides L from the plant through mowing in a large quantity.Sophora alopecuroides L is a kind of limited wild resource,however,now the irrational utilization of Sophora alopecuroides L.has led to deterioration of grassland.Artificial cultivation of Sophora alopecuroides L.costs a lot and the pesticide effect of the artificial cultivated Sophora alopecuroides L.is difficult to meet the demand.Endophytic fungi are commonly existed in plants.Many studies have shown that endophytic fungi can produce active ingredients,which effects are the same or similar as host plant.Based on this,this study was on the basis of detection system of Quinolizidine alkaloids of Sophora alopecuroides L.,use the isolated and purified fungi in the previous stage from Sophora alopecuroides L.as the material,filtrate the strains that producing quinolizidine alkaloids and then,identify them.To optimize the alkaloid-producing conditions by using response surface analysis of the effects of different factors on alkaloid synthesis by Sophora alopecuroides L endophytic fungi;using UV mutagenesis and nitrosoguanidine chemical mutagenesis to mutagenizing and select the mutant strains with high alkaloid production,to provide a theoretical basis for the study of alkaloid production mechanism of endophytic fungi of Sophora alopecuroides L and the exploitation of endophytic fungal resources of wild Sophora alopecuroides L.The results of the study are as follows.1.The best extraction method of Sophora alopecuroides L quinolizidine alkaloids was determined by using methanol as the extraction solvent and adding cellulase under the ultrasonic time of 60 min.A thin layer chromatography(TLC)method with ethyl acetate:ethanol:concentrated ammonia(5:1:0.5)as the best unfolding agent and modified potassium bismuth iodide as the color developer was established for the extraction and detection of Sophora alopecuroides L quinolizidine alkaloids,and a sample spotting volume of 5 μL was used for qualitative detection.The sample was detected qualitatively and quantitatively by high phase liquid chromatography(HPLC)with the mobile phase of 0.01 mol/phosphate buffer-methanol(55:45),UV detection at 216nm and a flow rate of 1.0mL·min-1.2.One locustine producing endophytic fungal strain DSD201 was obtained from 20 bitter bean seed endophytic fungi by alkaloid precipitation,TLC and HPLC methods,which was identified morphologically and molecularly as the fine pole streptomycete Alternaria alternata.3.The alkali-producing conditions of Sophora alopecuroides L endophytic fungus DSD201 were optimized,and the results showed that the strain DSD201 had the best mycelial growth with a dry weight of 0.971 g at 5 d in PDB medium,and the response surface analysis resulted in the addition of the precursor substance L-piperidinic acid at a concentration of 10×10-3.85 mol-L-1 and L-lysine at a concentration of 10×10-2.03 mol-L-1 and α-ketoglutaric acid at 10×10-4.31 mol-L-1,the maximum production of locustine by DSD201 strain was achieved.4.Using strain DSD201 as the material,one strain D2 with a significant increase in alkali production was screened by UV irradiation mutagenesis and chemical mutagenesis at treatment time of 20 min and a dose of 10 μL of nitrosoguanidine,the alkali production increased by 35.59%compared with the wild strain.At a UV irradiation time of 9 min and a lethality rate of 85.36%for strain DSD201,a strain E2 with a significant decrease in alkali production was selected,with a 47.88%decrease in alkali production compared to the wild strain.