Długoński J, Słaba M, Wilmańska D
Department of Industrial Microbiology, University of Lódź, Poland.
Microbios. 1998;95(381):71-7.
An electroporation method, utilizing cryopreserved protoplasts, has been developed for the steroid 11-hydroxylating fungus Curvularia lunata strain IM 2901. Protoplasts released from the apical parts of 24- and 48-h-old mycelia were suspended in cryopreservation buffer and stored at -75 degrees C for several weeks. The thawed and freshly prepared (control) protoplasts were electroporated with pAN 7-1 plasmid carrying the Escherichia coli hygromycin B resistance gene (hph) under the control of Aspergillus nidulans sequences. The electroporation efficiency of the control protoplasts with plasmid pAN 7-1 was 7.5 and 12.0 transformants per microgram DNA (protoplasts liberated from 24- and 48-h-old mycelia, respectively). Protoplasts released from the younger mycelium were more stable according to their reversion ability to mycelial form and transformation efficiency. After 16 weeks of cryopreservation the yield of electroporation was 61.3% of the control value. All isolated electrotransformants proved to be stable for a period of > 4 months even without selective pressure.
已开发出一种利用冷冻保存原生质体的电穿孔方法，用于甾体11-羟化真菌新月弯孢菌IM 2901菌株。将从24小时和48小时龄菌丝体顶端部分释放的原生质体悬浮于冷冻保存缓冲液中，并在-75℃下储存数周。将解冻后的原生质体和新鲜制备的（对照）原生质体，用携带在构巢曲霉序列控制下的大肠杆菌潮霉素B抗性基因（hph）的pAN 7-1质粒进行电穿孔。对照原生质体用质粒pAN 7-1的电穿孔效率分别为每微克DNA 7.5个和12.0个转化体（分别从24小时和48小时龄菌丝体中释放的原生质体）。根据从较年轻菌丝体释放的原生质体恢复为菌丝体形态的能力和转化效率，其更稳定。冷冻保存16周后，电穿孔产量为对照值的61.3%。所有分离的电转化体即使在没有选择压力的情况下，在超过4个月的时间内都证明是稳定的。
