Fostriecin产生菌Streptomyces pulveraceus遗传转化体系的建立与优化

Establishment and optimization of the conjugal transfer system of fostriecin producer Streptomyces pulveraceus

摘要:

【目的】建立并优化链霉菌Fostriecin产生菌Streptomyces pulveraceus的遗传转化系统。【方法】以整合型质粒pSET152为出发质粒, 通过供体菌E. coli ET12567/pUZ8002与受体菌Streptomyces pulveraceus进行接合转移。【结果】确定了链霉菌Streptomyces pulveraceus的最佳接合转移条件: 培养基为终浓度含15%甘氨酸的MS培养基; 孢子热激条件为50 °C 10 min; 阿伯拉霉素覆盖的时间为18 h, 终浓度为20 mg/L。同时, 把组成型启动子ermE+与绿色荧光蛋白基因(gfp)克隆到pSET152载体上, 通过接合转移整合到该链霉菌中, gfp获得表达。【结论】建立Fostriecin产生菌的遗传转化系统, 并发现甘氨酸能显著提高链霉菌的接合转移效率。

Abstract:

[Objective] Establishment and optimization of the gene transfer system of Streptomyces pulveraceus which produces fostriecin based on normal conjugal transfer method. [Methods] Intergeneric genetic transfer system was based upon integrative plasmid pSET152 from donor E. coli ET12567/pUZ8002 to Streptomyces pulveraceus. [Results] The maximum transformation efficiency of Streptomyces pulveraceus by conjugation was obtained when MS medium containing 15% glycine was used, with the spores heat-shocked at 50 °C for 10 min before mixing with E. coli, using a final 20 mg/L concentration of apramycin for overlay after incubation of 18 h. Simultaneity, pSET153::gfp was constructed from pSET152 by combining the constitutive ermE+ promoter with green fluorescent protein gene (gfp). Green fluorescent protein gene was expressed. [Conclusion] In summary, intergeneric genetic transfer system was demonstrated and optimized, and glycin can increase the efficiency of exconjugates.