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选用对5–氟乳清酸(5-FOA)敏感的金针菇单核菌株‘DG1-1’作为供试菌株,对其原生质体采用功率为10 W的紫外光进行垂直距离15 cm照射12 s的诱变处理,利用含有5-FOA和尿嘧啶的筛选培养基筛选获得了3株稳定的尿嘧啶营养缺陷型突变菌株‘NG1-65’、‘NG1-92’和‘NG1-95’。通过对尿嘧啶合成代谢路径中的pyrF和pyrG基因的分子检测发现,‘NG1-65’菌株的pyr F基因第39位和40位碱基之间插入T,‘NG1-92’菌株的pyrG基因第236位碱基T突变为C,‘NG1-95’菌株的pyrF基因第104位碱基C突变为T,这些插入突变和点突变可能导致基因编码的蛋白功能失去活性,产生尿嘧啶营养缺陷型。金针菇尿嘧啶缺陷型菌株可以为金针菇遗传转化体系的构建提供材料。
The mononuclear strain Flammulina velutipes ’DG1-1’ sensitive to 5-fluoroorotic acid (5-FOA) was selected as the test strain. The protoplasts were irradiated with UV light of 10 W at a vertical distance of 15 cm for 12 s Three stable uracil auxotrophic mutant strains (NG1-65 ’,’ NG1-92 ’and’ NG1-95 ’) were screened by screening medium containing 5-FOA and uracil. Through the molecular detection of the pyrF and pyrG genes in uracil metabolic pathways, it was found that the pyrG gene of NG1-92 ’was inserted between the 39th and 40th bases of the pyr F gene of NG1-65’ The 236th base T is mutated to C, and the 104th base C of the pyrF gene in the ’NG1-95’ strain is mutated to T. These insertion mutations and point mutations may result in loss of activity of the gene encoding the protein and uracil auxotrophy type. Flammulina velutipes uracil-deficient strains of materials for the construction of Flammulina genetic transformation system.