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 Micoromonosporaand Escherichia coli

作　　者：陈鑫[1] 杨振飞[1] 朱芮[1] 肖延辉[1] 郭利 王晓丽 李晓华[1] 

机构地区：[1]中南民族大学生命科学学院/生物技术国家民委重点实验室,武汉430074 [2]湖北省襄阳市烟草专卖局,襄阳441003

出　　处：《华中农业大学学报》2015年第4期80-83,共4页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31070087;30570046);湖北省自然科学基金重点项目(2011CDA079);湖北省烟草公司面上项目(0710XYYC2012-01);国家大学生创新创业训练项目(GCX13104);"十二五"中南民族大学国家级民族药学实验教学中心建设项目

摘　　要：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4.7kb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40μL和400μL。To construct the conjugation transferring system between rare actinomycetes Micoromonosporaand E.coli which can transfer foreign genes into Micoromonospora,a 4.7kb 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 pUZ8002and subsequently transferred by conjugation into Micoromonosporasp.LXH20 with selection of apramycin-resistant colonies,exconjugants were obtained. The plasmid pSCU207 was introduced by conjugation into Micromonosporasp.LXH20 and stably maintained in Micromonosporasp.LXH20.The optimal conjugation volume of E.coli ET12567 carrying pSCU207and Micromonosporafresh mycelium was 40μL and 400μL,respectively.

关 键 词：小单孢菌 接合转移 质粒pSCU207 抗生素的敏感性 质粒稳定性 

分 类 号：Q933[生物学—微生物学]