稀有放线菌小单孢菌与大肠杆菌接合转移体系的构建
Constructing the conjugation transferring system between rare actinomycetes Micoromonospora and Escherichia coli

为将外源基因导入小单孢菌，建立小单孢菌（Micromonospora）与大肠杆菌两亲接合转移方法，将包含小单孢菌内源质粒pJTU112复制区的4.7 kb片段插入质粒pOJ260的BamHⅠ酶切位点，构建了能在大肠杆菌和小单孢菌中复制的穿梭载体pSCU207，将质粒pSCU207转化大肠杆菌ET12567/pUZ8002，再与小单孢菌LXH20进行两亲接合转移，挑取接合转移子，并进行验证。结果表明：质粒pSCU207通过大肠杆菌与小单孢菌新鲜菌丝之间接合转移导入小单孢菌中，并可稳定遗传；大肠杆菌与小单孢菌新鲜菌丝之间的最佳接合转移体积是40 μL和400 μL。
To construct the conjugation transferring system between rare actinomycetes Micoromonospora and E. coli which can transfer foreign genes into Micoromonospora, a 4.7 kb fragment containing the replication region of plasmid pJTU112 was cloned into the BamHⅠ site of vector pOJ260 which can be replicated in both E. coli and Micromonospora. The plasmid pSCU207 was introduced into E. coli ET12567 carrying pUZ8002 and subsequently transferred by conjugation into Micoromonospora sp. LXH20 with selection of apramycin-resistant colonies,exconjugants were obtained. The plasmid pSCU207 was introduced by conjugation into Micromonospora sp. LXH20 and stably maintained in Micromonospora sp. LXH20. The optimal conjugation volume of E. coli ET12567 carrying pSCU207 and Micromonospora fresh mycelium was 40 μL and 400 μL,respectively.