利用弗氏链霉菌的修饰系统克服吸水链霉菌的限制性障碍——发展吸水链霉菌转化系统的尝试

Overcoming Restriction of Streptomyces Hygroscopicas 10-22 by the Modification of S. fradiae——An Attempt to Develop a Transformation System for S. hygroscopicus 10-22

用来自变铅青链霉菌TK24的质粒plJT02(tsr、mel^+)转化吸水链毒菌应城变种10-22的原生质体,未能得到转化子。但改用来自弗氏链霉菌ATCC 10745的plJ702转化10—22原生质体却得到了转化子。转化频率约10 ~3—10~4转化子/微克DNA。在这些转化子中,只有约1/1000是黑色菌落(mel^+),绝大部分为白色菌落(mel^-)。分别挑出黑色、白色2种菌落,只有从前者能提出电泳可见的plJ702质粒带。2种菌落的质粒提取物,均能转化TK24原生质体并正常表达黑色素。在非选择性条件下,从10-22(pIJ702)黑色菌落的群体中,获得了少数白色菌落,经证明它们是10-22的宿主突变体。这些突变体的plJ702转化子出现均一的黑色菌落。

No transformant was obtained when pIJ702 (tsr, met+) from S. lividans TK24 was used to transform S. hygroscopicus 10-22. pIJ702 isolated from S. fradiae ATCC 10745, however, was transformed into 10-22 at a frequency of 108-104 transformants/μg DNA. Among the transformant colonies, only 1/1000 of them were black in colour (mel+) while a great majority of them remained white (mel-). Plasraid plJ702 band was only visualized on agarose gels from the black colonies but not from the white colonies. However, when plJ702 isolated from both black and white transformants were used to transform S. lividans TK24, the mel gene was expressed normally in the recipients. The preparations was also successful in transforming S. hygroscopicus 10-22, and again gave rise to 1/1000 of black colonies only. When the 10-22 (plJ702) black colonies were plated on non-selective medium, among the majority of black colonies grown, there were a few white colonies, which were proved to be host mutants of 10-22. These mutants were transformable by pIJ702 and homogenous black colonies were obtained.