Yanai K, Yonekura K, Usami H, Hirayama M, Kajiwara S, Yamazaki T, Shishido K, Adachi T
Bio Science Laboratories, Meiji Seika Kaisha, Ltd., Saitama, Japan.
Biosci Biotechnol Biochem. 1996 Mar;60(3):472-5. doi: 10.1271/bbb.60.472.
A plasmid pLC-bar containing the bialaphos resistance gene derived from Streptomyces hygroscopicus between the Lentinus edodes ras gene promoter and priA gene terminator was constructed. When protoplasts of Pleurotus ostreatus were mixed with the plasmid DNA in the presence of polyethylene glycol and CaCl2, bialaphos-resistant colonies were obtained. This indicated that transformation was successful. Southern blot analysis of total DNAs from transformants showed that the introduced plasmid DNA was integrated into the host chromosome and partly rearranged. A plasmid, pLC-GUS, containing the Escherichia coli beta-glucuronidase (GUS) gene under the control of the L. edodes ras gene promoter and priA gene terminator was constructed and introduced into protoplasts of P. ostreatus with pLC-bar by co-transformation. Two of 5 transformants obtained as bialaphos-resistant colonies showed two to twenty times higher specific activity of GUS than the recipient. Southern blot analysis of total DNAs from transformants indicated the presence of the GUS gene only in the two transformants. These results indicated that co-transformation of P. ostreatus was successful, and that the GUS gene was expressed in P. ostreatus. This transformation system will enable us to breed commercial strains of P. ostreatus at the molecular level.
构建了一种质粒pLC-bar，其在香菇ras基因启动子和priA基因终止子之间含有来源于吸水链霉菌的双丙氨膦抗性基因。当在聚乙二醇和氯化钙存在的情况下，将糙皮侧耳原生质体与质粒DNA混合时，获得了双丙氨膦抗性菌落。这表明转化成功。对转化体的总DNA进行Southern印迹分析表明，导入的质粒DNA整合到宿主染色体中并发生了部分重排。构建了一种质粒pLC-GUS，其在香菇ras基因启动子和priA基因终止子的控制下含有大肠杆菌β-葡萄糖醛酸酶（GUS）基因，并通过共转化将其与pLC-bar一起导入糙皮侧耳原生质体中。作为双丙氨膦抗性菌落获得的5个转化体中有2个显示出比受体高2至20倍的GUS比活性。对转化体的总DNA进行Southern印迹分析表明，仅在这两个转化体中存在GUS基因。这些结果表明糙皮侧耳的共转化成功，并且GUS基因在糙皮侧耳中表达。该转化系统将使我们能够在分子水平上培育糙皮侧耳的商业菌株。
