海洋链霉菌分离株M095遗传转化体系的建立

研究了海洋链霉菌分离株M095的基因转移系统。利用属间接合转移将具有oriT的大肠杆菌-链霉菌穿梭质粒pIJ8600转入EscherichiacoliET12567(pUZ8002)中,获得供体菌。将供体菌与预萌发的菌株M095的孢子进行接合转移,将质粒pIJ8600转入菌株M095中,其转化率为1.99×10-4个接合转化子/受体。Southern杂交证明质粒pIJ8600已经整合到菌株M095的染色体上。同时,将来自Spirulinamaxima(Cyanophyta)的别藻蓝蛋白基因(apc)克隆在质粒pIJ8600的XbaI和BglII位点,产生质粒pAPIJ。用接合转移法将质粒pAPIJ转入菌株M095。通过SDS-PAGE分析,得到2个大小为22ku和17ku的蛋白,分别相对应于别藻蓝蛋白的α和β亚基。这些结果进一步证明菌株M095的遗传转化体系已经成功建立起来,这将为其他海洋放线菌遗传转化工作的研究奠定基础。

Gene transfer system for the marine Streptomyces sp. isolated M095

We established a gene transfer system for marine Streptomyces sp. M095. A recombinant Escherichia coli ET12567(pUZ8002, pIJ8600) was obtained by transforming E. coli ET12567(pUZ8002) with oriT-containing E. coli-Streptomyces shuttle plasmid pIJ8600. In conjugal transfer experiments, E. coli ET12567(pUZ8002, pIJ8600) was the donor, and the recipient was marine Streptomyces sp. M095 spore after pregerminating by heat shock. Mating between donor and recipient was conducted. Plasmid pIJ8600 was introduced into strain M095 by conjugantion from E. coli ET12567. The transformation frequency for this system was approximately 1.99×10^-4 exconjugants/recipient. Southern hybridization analysis indicated that plasmid pIJ8600 integrated at a unique site in the chromosome of marine Streptomyces sp. M095. In addition, the Allophycocyanin gene(apc) originating from Spirulina maxima(Cyanophyta) was cloned between XbaI and BglII sites of pIJ8600, yielded the plasmid pAPIJ. Plasmid pAPIJ was transferred into marine Streptomyces sp. M095 by conjugation, which was used to demonstrate this transformation system. By SDS-PAGE analysis, two proteins with molecular masses of 22 and 17 ku, which were corresponding to the alpha and beta subunits of APC, were expressed in strain M095/pAPIJ. These results indicated that a genetic transformation system for marine Streptomyces sp. isolated M095 was successfully established.