Screening For The Claviceps Purpurea And Analysis Of The Metabolites Of The Claviceps Purpurea

Ergot is caused by Claviceps species that has been found on hundreds of plants, Claviceps purpurea is the most widely known species. The ergot alkaloids in ergot are a complex family of indole-derived with varied and important biological activities. These alkaloids are used to treat migraines, Parkinson’s disease, prolactinomas and reduce bleeding during child birth, ect. Claviceps purpurea, Claviceps fusiformis and Claviceps paspali are producing strains of ergot alkaloids, they can produce different alkaloids. Claviceps fusiformis can produce lysergic acid derivatives and Claviceps paspali is used to produce clavinet alkaloids, whereas only Claviceps purpurea produces peptide alkaloids. At present there are few researches on Claviceps purpura in our countries, and the ergot fungus used in the research were mostly obtained from foreignal countries.We have studied the effective components in rye sclerotias of Tibetan Pastoral Areas, Ukraine and Russia. Through TLC and ESI-MS, we inferred that these sclerotias contain peptide alkaloids.This research firstly screened ergot alkaloids producing strains from rye sclerotias of Tibetan Pastoral Areas, Ukraine and Russia. Using the method of shake flask fermentation, we finally screened38strains that have alkaloids producing ability from305strains. We found a purple strain on A50plate, and then this purple strain was used for studying the genetic stability of purple character and stability of alkaloids producing. At the end, a purple strain with stable alkaloids producing ability was obtained, and the strain was munbered A50-1.Simlilarity analysis based on26S rDNA D1/D2domain sequences and the sequence of ITS region were used to identify the isolated strain, and the result indicted that the strain isolated was Claviceps purpurea.We have isolated three compounds from the metabolites of A50-1. After a series of ananlysis by color reaction, TLC, ESI-MS and1HNMR, we confirmed zp-2-160-2was ergometrine and zp-2-12-1was ergocristine and we also speculated the chemical structure of zp-2-160-1. Subsequently, A50-1was used to investigate the fermentation condition. The result showed that the optimal seed medium was M2, and the optimal fermentation mediun was SC.A50-1was treated by60Co, Six strains with different morphology were also obtained through the mutation. Study on these six strains, we detemined the relationship between morphology of strain and the kind of ergot alkaloids.We analysed these strains by the method of RAPD. In experiment, factors which may affect the result of RAPD and the program of RAPD-PCR were optimized. Finally, we determined the optimal conditions of RAPD-PCR. Based on the result of RAPD-PCR, we calculated the similarity coefficient and a dendrogram was constructed by using UPGMA’s method.