The Mycelium Artificial Cultivation And The Toxin Detection Of Two Kinds Galerina

Amanita toxic peptides are extremely poisonous. Their mechanism is inhabiting the activity of the eukaryotic RNA polymerase Ⅱ,which promote the development of medical biology, molecular biology, cell biology, biological developmental and other disciplines. Most of the Amanita toxic peptides exist in some Amanita mushrooms and Galerina mushrooms. As ectomycorrhizal fungi, Amanita mushrooms are difficult to cultivate artificially. Therefore, the amanita toxic peptides are mainly extracted from wild Amanita mushrooms at present; but because of the limited resources, the prices of amanita toxic peptides are expensive. As saprophytic fungi, the cultivation of Galerina mushrooms is relatively easy. In this paper, we cultured the mycelium of Galerina mushrooms, and detected the amanita toxic peptides of their fruiting body and mycelium.The experiment materials of this article described are collected in Hunan Liuyang, Hunan Suining, Hunan Baojing and Xinjiang Wulumuqi. We combined traditional forms with modern ITS sequence alignment methods to identify species, found that there are two specimens in the four place; Among them, the species come from Hunan Liuyang, Suining, Baojing, are Galerina sulciceps(Berk.) Singer; and the species come from Xinjiang are Galerina marginata(Batsch) Kuhner. Through screening, we selected the specimen from Liuyang and Sinkiang as materials for subsequent experiments.In this paper, we isolated two strains throughing spores and tissue isolation method. Based on this, we start the pure culture of mycelium. Further more, we sequenced the mycelia and fruiting body of the two species, then got and compared ITS sequence, we found that the ITS sequence in mycelia and fruiting body of corresponding Galerina mushroom are identical. So we firmly believe that separated myceliums are come from the corresponding Galerina mushrooms. Through screening the medium, determining the optimal basic medium, and through the single factor screening, including carbon sources, nitrogen sources, pH and temperature, we determined the optimal culture conditions of the two strains, respectively. Finally, we extracted toxins in fruiting body and mycelium of the two species respectively. Using HPLC testing, we determined the kind of amanitin and toxin content.By solid medium screening, we determined HSVA as the optimum foundation culture medium. Through the single factor screening, found the optimal nitrogen source of the two species are peptone(1g/L)+ NH4CL(0.1g/L), the optimum carbon source of them is glucose, and the G. marginata demand for glucose is a little higher than G. sulciceps. The optimal temperature of them are 20 ℃. Their pH value application scope are wide. By liquid medium screening, we found the optimal carbon source of G. sulciceps is glucose, and the optimal carbon source of G. marginata is brown sugar. Through HPLC analysis and detection, we found that the α-Ama content in fruiting body of G. sulciceps is 1795.3μg Per gram of dry weight, and the β-Ama content in fruiting body of G. sulciceps is 1925.0μg Per gram of dry weight. The α-Ama content in fruiting body of G. marginata is 445.4μg Per gram of dry weight, and the β-Ama content in fruiting body of G. marginata is 527.0μg Per gram of dry weight. Then, we detected amanita toxic peptides in the mycelia and fermented liquid of G. sulciceps and G. marginata.The biggest innovation point of this article is that, in domestic, we screened the optimal culture conditions of G. sulciceps and G. marginata, and detected amanita toxic peptides in fruiting body and mycelia of them for the first time.