大肠杆菌-链霉菌穿梭表达载体pMF的构建及其应用  

Construction of Escherichia coli-Streptomyces shuttle expression plasmid pMF

作　　者：全梅芳[1] 余凯[1] 夏立秋[1] 丁学知[1] 曾凡军[1] 寇笑笑[1] 王海龙[1] 胡胜标[1] 余子全[1] 李敬业[1] 

机构地区：[1]湖南师范大学生命科学学院,微生物分子生物学湖南省重点实验室

出　　处：《微生物学报》2010年第9期1251-1257,共7页Acta Microbiologica Sinica

基　　金：国家“863计划”(2006AA02Z187,2006AA10A212);国家自然科学基金(30870064,30670052);教育部博士后基金(20060542006);湖南省教育厅资助项目(04C381)~~

摘　　要：【目的】构建能定点整合到链霉菌（Streptomyces）染色体上的高效表达载体。【方法】以链霉菌自杀型表达载体pLSB2为基础,通过插入链霉菌噬菌体ΦC31整合酶基因int和attP位点（Phage attachment site）,构建了能在大肠杆菌和链霉菌之间进行接合转移并定点整合到链霉菌染色体上的表达载体pMF。将pMF转化大肠杆菌ET12567（pUZ8002）,并分别接合转移天蓝色链霉菌（Streptomyces coelicolorM145）、变铅青链霉菌（Streptomyces lividansTK24）和红色糖多孢菌（Saccharopolyspora erythraea2338）,挑取接合子进行PCR和Southern杂交检测。将来自刺糖多孢菌S08-4的S-腺苷甲硫氨酸合成酶基因（SAM-s）克隆到载体pMF的启动子下游,接合转移到天蓝色链霉菌中。【结果】表明pMF成功整入链霉菌染色体,并且检测到目的蛋白的表达。【结论】构建的pMF载体可作为外源基因定点整合表达的有效工具,为后续的基因功能研究以及链霉菌的遗传改造奠定了基础。[Objective]To construct an E.coli-streptomyces shuttle vector pMF that can integrate into the genome of streptomyces by site-specific integration.[Methods]We inserted the integrase gene ΦC31 int and attP site into pLSB2,a suicidal streptomyces plasmid.The resulting conjugably transferable vector which contains the activator promoter system act Ⅱ-ORF4/PactⅠfrom Strepromyces coelicolor A3（2） could be integrated into the genome of streptomyces by site-specific integration.[Results] The plasmid pMF was conjugably transferred with high frequency into S.coelicolor M145,S.lividans TK24 and Saccharopolyspora erythraea 2338 from E.coli.Southern blotting results showed that pMF was able to integrate into the genome of streptomyces.We also confirmed functional protein expression by cloning a putative Sadenosylmethionine synthetase（SAM-s） gene from Sacc.spinosa S08-4 into pMF and conjugated into S.coelicolor M145.Protein expression were confirmed using Western blotting.[Conclution]pMF can be used as an effective tool for sitespecific integration expression of foreign gene in streptomyces.

关 键 词：链霉菌 整合酶 表达载体 接合转移 

分 类 号：Q78[生物学—分子生物学]