大肠杆菌-链霉菌高效接合载体的构建及其应用

Construction of Efficient Conjugal Plasmids Between Escherichia coli and Streptomycetes

以链霉菌质粒SCP2 的衍生质粒pHJL4 0 0为基础 ,构建了能够在大肠杆菌到链霉菌之间进行高效接合转移的质粒pGH112。pGH112含有在大肠杆菌和链霉菌中复制起始位点 ,以及分别在大肠杆菌和链霉菌中进行筛选的抗性标记。用pGH112转化EscherichiacoliET12 5 6 7(pUZ80 0 2 )后 ,与天蓝链霉菌 (StreptomycescoelicolorA3(2 ) )、除虫链霉菌 (Streptomycesavermitilis)、变铅青链霉菌 (StreptomyceslividansTK5 4 )、毒三素链霉菌 (StreptomycestoxytriciniNRRL15 4 4 3)、委内瑞拉链霉菌 (Streptomyces.venezuelaeISP5 2 30 )和红色糖多孢菌 (Saccharopolyporaerythraea)进行接合 ,发现本文构建的pGH112与pKC1139相比 ,接合转移效率较高 ,稳定性好 ,而且宿主范围较广。把组成型启动子ermE 与绿色荧光蛋白基因 (gfp)克隆到本文构建的pGH112 ,通过接合转移到链霉菌中 ,gfp获得表达 ,证明其可以用作基因接合转移的有效工具载体 。

Conjugal plasmid pGH112 has been developed based on the replicons of Streptomyces coelicolor plasmid SCP2 * and E.coli ColE. The plasmid contains ampicilin resistance gene( amp ) for selection in E.coli and thiostrepton resistance gene ( tsr ) for selection in Streptomycetes, and a 0.76 kb oriT fragment of (IncP) RK2. Conjugal transfer of pGH112 was performed from E.coli to S.coelicolor A3(2), S.avermitilis , S.lividans TK54, S.toxytricini NNRL15443, S.venezuelae ISP5230 and Sacc.erythraea by conjugation, results show that the plasmid was able to transfer efficenctly from E.coli to Streptomycetes, was stably inherited in the recipients. pGH113 was constructed from pGH112 by combining the constitutive erm E * promoter with green fluorescent protein gene( gfp ).