电转化法将luxAB基因导入毒死蜱降解菌β菌株的研究

luxAB labelling of chlorpyrifos-degrading strain β by electroporation

采用电转化法将带发光酶基因luxAB的质粒pTR102导入毒死蜱降解菌β菌株,研究了细胞生长状态、电压强度、电击时间和质粒浓度对转化效率的影响,并对转化子的生理特性进行研究.实验结果表明,以处于对数生长早期的菌液制备感受态细胞,在质粒浓度16.5μg·mL-1、电压2.5kV,电击时间3ms条件下的转化率最高,可达2.73×102个.μg-1(以每μg质粒DNA中所含转化子个数计).转化子lux-β分别在抗生素平板和LB平板中传代10次,仍具有发光活性和对卡那霉素(Km)的抗性,说明质粒pTR102在转化子中可以稳定遗传.转化子的生理特性变化不显著,对毒死蜱的降解能力较出发菌株提高了近1倍.

A new luxAB gene-labelling method was established in this study in order to provide a method of investigating and tracing the colonization dynamics and degradation activity of chlorpyrifos-degrading bacteria in the natural environment. The luxAB genes were introduced into chlorpyrifos-degrading strain β by electroporation. Cells in initial logarithmic phase were more sensitive to foreign DNA,and the maximum transformation rate was up to 2.73×10^2 transformants per microgram plasmid DNA when the plasmid concentration was 16.5μg·mL^-1 under the conditions of voltage 2.5 kV and time 3 ms. After plate transfers with or without antibiotics for 10 generations the transformant lux-β still had luminescence activity and Km resistance,indicating that the transformant was genetically stable. In addition,the physiological and biochemical characteristics of lux-β changed little,and the chlorpyrifos-degradation rate of lux-β doubled compared with that of the wild strain β.