鼠李糖乳杆菌电转化条件的研究

Studies on the Electro-transformation Conditions of Lactobacillus rhamnosus

目的:研究不同因素对鼠李糖乳杆菌电转化效率的影响,探讨电转化的最适条件,提高电转化效率。方法:以鼠李糖乳杆菌05-28为受体菌株,大肠杆菌-乳酸菌穿梭型质粒p MG36e为载体,通过原生质体电转化的方法研究了质粒浓度、电击缓冲液、电转化参数、复苏培养基组成及复苏培养时间对电转化效率的影响。结果:利用1μL质量浓度为1μg/μL的质粒p MG36e,在电场强度8 k V/cm,电阻200Ω,电容25μF的条件下进行电击转化,以含有0.5 mol/L蔗糖和20 mmol/L的MgCl_2、CaCl_2且无吐温80的MRS再生培养基复苏培养2.5 h,并采用添加红霉素的筛选培养基,获得1.66×105CFU/μg DNA的电转化效率。结论 :实现了鼠李糖乳杆菌的高效遗传转化,为遗传育种提供了技术支持。

Objective： Different factors affecting electro-transformation efficiency of LactobaciUus rhamnose were studied and optimal condition of electro-transformation were selected to improve the transformation efficiency. Methods： Lacto- bacillus rhamnose was used as a host, and the E. coli - lactic acid bacteria shuttle plasmid pMG36e were used as a vector; through the method of protoplast electro-transformation, the conditions needed for transformation efficiency were studied, which included plasmid concentration, electric shock buffer, electro-transformation parameters, composition of the recovery culture and incubation time of recovery. Results： Using 1 μL concentration of 1 μg/μL plasmids pMG36e, with the electroporation parameters of 8 kV/cm, 200 Ω and 25 μF, and proper incubation in MRS broth containing 20 mmol/L MgCl2, CaCl2 and sucrose of 0.5 mol/L （without Tween 80） for at least 2.5 hours, the best electroporation effi- ciency was obtained. As a result, the transformational efficiency reaches 1.66×10^5 CFU/μg DNA. Conclusion： This study has realized efficientgenetic transformation of Lactobacillus rhamnosus and provided the technical support for genetic breeding.