链霉菌质粒pSET152电转化稀有放线菌小单孢菌的研究

Study on electroporation of rare Actinomycete Micromonospora sp.40027 with Streptomyces plasmid pSET152

摘要:

利用链霉菌(Streptomyces)噬菌体ΦC31所构建的整合型载体pSET152作为供体质粒,分别以小单孢菌(Micromonospora)40027菌株的萌发孢子和新鲜菌丝体作为受体菌,在不同的电场强度下进行电转化实验,结果表明:以小单孢菌40027菌株萌发孢子为受体菌,未获得电转化子;以小单孢菌40027菌株新鲜菌丝体为受体菌,获得了电转化子。电场强度为13kV/cm时可获得最高转化效率。Southern杂交结果表明:质粒pSET152可通过菌丝体电转化法导入小单孢菌40027菌株,并整合到小单孢菌40027菌株的染色体上,暗示链霉菌噬菌体ΦC31的整合酶基因和整合位点在异源宿主小单孢菌40027菌株中仍具有相同的功能。质粒稳定性检测实验表明:质粒pSET152可稳定地存在于小单孢菌40027菌株中。

关键词:小单孢菌40027菌株; 电转化; 质粒

Abstract:

Electroporation of Micromonospora sp.40027 isolated from soil was studied with Streptomyces plasmid pSET152, an integrative vector commonly used in Streptomyces genetic manipulation. Transformant was not obtained by electroporation with germinated spores of Micromonospora sp.40027 as recipient, but plasmid pSET152 can be electroporated into the fresh mycelium of Micromonospora sp.40027, and the highest electroporation efficiency was yielded under the electric field strength of 13kV/cm. Plasmid stability experiment and southern blot showed that pSET152 could stably exist in the Micromonospora sp. 40027, and was integrated into its chromosome via the attP site, originated from Streptomyces phage C31. These data suggested that plasmid pSET152 was successfully electroporated into Micromonospora, and that the integrase gene and attP site of Streptomyces phage ΦC31 could play the same role in Micromonospora.

Key words:Micromonospora sp. 40027; electroporation; plasmid