金针菇尿嘧啶营养缺陷型菌株的筛选与分子鉴定  

Screening and Molecular Verification of Uracil Auxotrophic Mutants of Flammulina velutipes

作　　者：卢绪志 万佳宁[1] 茅文俊[1] 杨瑞恒[1] 王瑞娟[1] 鲍大鹏[1] 

机构地区：[1]上海市农业科学院食用菌研究所,农业部南方食用菌资源利用重点试验室,国家食用菌工程技术研究中心,上海市农业遗传育种重点开放试验室,上海201403 [2]上海海洋大学食品学院,上海201306

出　　处：《园艺学报》2017年第3期495-503,共9页Acta Horticulturae Sinica

基　　金：上海市科学技术委员会工程技术研究中心能力提升项目(16DZ2281300);上海市农业科学院科技发展基金项目(沪农引才2016第001号)

摘　　要：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。Flammulina velutipes is one of the most popular edible mushroom species in the world.In this study,3 uracilauxotrophic mutants‘NG1-65’,‘NG1-92’and‘NG1-95’were obtained from the monocaryon 5-fluoroorotic acid（5-FOA）-sensitive F.velutipes strain‘DG1-1’by UV irradiation.UV mutagenesis was conducted under the conditions as following：an irradiation intensity of 10 W,irradiation distance of 15 cm and irradiation time of 12 s.The mutants could grow well in MM media containing 0.05 mmol · L^（-1） uridine and 0.5 g · L-1 5-FOA,but could not grow on MM medium without uridine.PCR analyses revealed that two critical genes pyr F and pyr G in the uridine biosynthetic pathway were disrupted in mutants,probably resulting in the inactivation of related proteins.The survey of the mutation sites suggested that a‘T’inserted the site between the 39 th and 40 th base of pyr F in strain‘NG1-65’and the 104 th base‘C’was changed into‘T’of pyr F in strain‘NG1-95’.The mutant T→C at the site of 236 th base was critical to inactive of pyr G in strain‘NG1-92’.The uracil auxotrophs of F.velutipes could be used as a tool for genetic transformation study and strain source identification.

关 键 词：金针菇 尿嘧啶营养缺陷型 pyrF基因 pyrG基因 

分 类 号：S646.15[农业科学—蔬菜学]