质粒pIJ702转化天蓝淡红链霉菌SIPI1482菌种的条件研究

STUDY ON TRANSFORMATION OF PLASMID pIJ702 INTO STREPTOMYCES COERULEORUBIDUS SIPI 1482

天蓝淡红链霉菌ＳＩＰＩ１４８２是一株蒽环类抗生素柔红霉素的产生菌。在建立该菌种基因工程研究所需的载体宿主系统的研究中，发现从变青链霉菌分离的质粒ｐＩＪ７０２不能直接转化ＳＩＰＩ１４８２菌种。经排除ＳＩＰＩ１４８２菌种自身内含质粒，试验了热休克衰减ＤＮＡ酶活性、不同时间培养的菌丝体制备的原生质体、ＰＥＧ１０００的浓度以及硫涟丝菌素选择时间等因素对ｐＩＪ７０２转化ＳＩＨＩ１４８２菌种的影响，证实上述绪因素都不是决定是ｐＩＪ７０２转化ＳＩＰＩ１４８２菌种成败的主要原因。采用ＳＩＰＩ１４８２菌种的阻断突变株Ｎ－１８为中介宿主菌，最终获得了质粒ｐＩＪ７０２转化ＳＩＰＩ１４８２菌种的成功。从Ｎ－１８转化子分离的质粒ｐＩＪ７０２再转化Ｎ－１８和亲株ＳＩＰＩ１４８２，观察到转化频率的大幅度提高，这些结果表明ＳＩＰＩ菌株具有限制一修饰系统，采用突变株为中介宿主可能是克服质粒转化限制性障碍的一个有效方法。

An attempt was made to introduce plasmid pIJ702 into S.coeruleorubidus SIPI 1482 which is a Daunomycin producer,however,it wasfound that plasmid pIJ702 isolated from Streptomyces lividans failed totransform the protoplasts of S. coeruleorubidus SIPI 1482.Other conditions for transformation,such as elimination of endogenic plasmid,attenuation ofDNase,age of mycelium,concentration of PEG1000 and time for thiostr-epton selection were also tested,but they were helpless to the transforma-tion of plasmid pIJ702 into strain SIPI 1482,when blocked mutant N-18 wastransformed with the same lasmid.a few transformants were found on a R2YE plate. It indicated that pIJ702 was certainly introduced into the pro一toplasts of mutant N-18 though the frequency of transformation was verylow.A much higher frequency of transformation was observed when themodificd plasmid pIJ702 isolated from transformants of N-18 was used toretransform mutant N-18 and its parent strain SIPI 1482.It implied thatrestriction and modification systems occured in S,coeruleorubidus SPIP 14 82.