Screening And Analysis Of The Polyorphism For The Claviceps Paspali

The ergot fungus of the family Clavicipitaceae is the main producer of ergot alkaloids, Claviceps paspali is one of the most important fungus and its major metabolites are ergoamides alkaloids. Due to the important role of ergot alkaloids in toxicology and pharmacology, it is widely used in modern medicine. Ergot alkaloids interact with serotonin, dopamine receptor and adrenergic receptor in the central nervous system. At present, ergot alkaloids were used in the treatment of postpartum hemorrhage, antihypertension, Parkinson’s disease and inhibition the secretion of pituitary hormone. Nowdays, because of lacking ergot resources in our country and the seriously degradation of ergot fungus, it’s difficult to obtain ergot alkaloids from large scales of the industry fermentation. As a result, the supply of ergot drugs still depend on import.The Claviceps paspali strain was preserved in our laboratory, and used as the research material for selection and breeding. We set up a high-throughput screening method for Claviceps paspali. Using 24 well plates and shake flask screening method, we obtained the strain C6 with high yield and high stability. The production of ergot alkaloids of strain C6 was 101.33 μg/mL.We used strain C6 as the starting strain for chemical mutation breeding and chose acridine orange, dimethylsulfate and N-methyl-N’-nitro-N-nitrosoguanidine as mutagens. After mutation breeding,120 single colonies were selected for high-throughput screening method with 24 well plates. In results, using N-methyl-N’-nitro-N-nitrosoguanidine mutation breeding, we screened a spore-producing strain C6-28. The strain C6-28 was higher yield and better stability, compared with strain C6, the yield of ergot alkaloids was increased by about 50%.We studied the seed and fermentation conditions of strain C6-28, which including optimizate the fermentation conditions with Plackttt-Burman experiment. In this study, the optimum seed growth time, the amount of spore inoculation, the amount of seed inoculation and the optimum fermentation time were determined, and selected M medium as the seed medium and chose M2 medium as the fermentation medium.In this study, the mutagenic material was the spores of strain C6-28. We counted the spores to achieve the effect of quantitative mutation. After four times of MNNG mutagenesis breeding, we obtained a high-yielding strain B3-24 and the yield of ergot alkaloids was 815.5 μg/mL. Compared with the starting strain C6, it was increased by about 8 times. The mycelium of strain B3-24 was characterized as rough shape, segmented obviously and large mounts of moniliform hyphae, the above characteristics were the same as the morphological characteristics of high yield strains. The results showed that using the spores of Claviceps paspali for MNNG mutagenesis was effectivity.Five different morphology mutant strains were obtained from the mutation breeding of strain F33. We utilized the improved benzyl chloride method to extract DNA of the six different morphology strains. Using RAPD technology to analyze the polymorphism of these strains. Based on the RAPD amplification results to construct a cluster analysis dendrogram, and further study on the differences between these strains from the gene level.