用电脉冲法将外源质粒DNA导入花生根瘤菌的研究

Study on the Introduction of Exogeneous Plasmid DNA into Penaut Rhizobia by Electroporation

利用基因电转移仪（GenePulserTM），对快、慢生型花生根瘤菌85－7和147－3的电转化条件进行了系统的研究。结果表明：在对数中期（ABC600为0．6－0．7）收获的细胞在最高场强（12．5kV／cm）和短脉冲时间（2．5－5．0msec）时达到最高转化效率（105转化子／μgDNA）．转化子数随DNA终浓度在一定范围（26．24Pg－1．5μg／ml）内呈线性增加，随即表现出“饱和效应”。增加受体菌细胞浓度，能提高电转化效率；而质粒分子量的增加（11．9－166kb）却使电转化效率降低。来自受体菌自身的同源质粒，因克服了宿主的限制一修饰性，可以极显著地提高电转化效率。电脉冲处理对受体菌自发突变没有影响。

A systematic study of transformation conditions of peanut rhizobia 85-7 and 147-3 was con-ducted by using Bio-Rad Gene Pulser equipment. It was rcvealed that the highest transformation efli-ciency (105 transformants / μg DNA) was obtained from cells harvested at mid-log phase of ABC600 on 0.6-0.7 under the highest field strength (12.5kV / cm) and a short pulsc length (2.5-5.0msec). A linearincrease of transformants was observed when DNA concentration was increased in the range of 26.24pg -1.5μg / ml and it bccame saturation afterwards. Transformation efficiency was also increased with the raise of recipient cell conccntrations, but decreased with the increase of plasmid sizes from 11.9 to 166kb.A significant increase of transformation efficiency was revcaled with the homologous plasmid isolated from recipient itself since the effects of host restriction and modification werc avoided. No significant ef-fect of electroporation on spontaneous mutation was observed.