改良醋酸锂转化法在白念珠菌质粒转化中的实验研究

A study of modified lithium acetate transformation for plasmid transformation in Candida albicans

目的:通过增加白念珠菌细胞壁通透性改良传统醋酸锂转化法的质粒转化率。方法:重组质粒经酶切线性化后,分别检测不同二甲基亚砜(DMSO)浓度(1%、5%和10%)化学处理和热休克时间梯度(0.5、2.0、3.0和4.0 h)白念珠菌的质粒转化率,筛选最适宜DMSO浓度及热休克时间,构建改良醋酸锂转化法。随后,采用选择性培养法和PCR验证比较传统醋酸锂转化法及改良醋酸锂转化法白念珠菌的质粒转化率。结果:发现采用5%的DMSO化学处理和将42℃热休克时间调整为3 h后阳性克隆子数量增多最明显。改良醋酸锂转化法的的白念珠菌质粒转化率为1.5×105阳性克隆子/1μg质粒DNA/108个细胞,而传统醋酸锂转化法转化率为0.6×105阳性克隆子/1μg质粒DNA/108个细胞。改良醋酸锂转化法的转化率明显高于传统醋酸醋酸锂转化法,两种方法质粒转化率的统计学比较存在显著性差异。结论:增加二甲基亚砜化学处理和调整热休克时间的改良醋酸锂转化法可以显著提高白念珠菌质粒转化率。

Objective：Based on the traditional transformation methods of polyethylene glycol/lithium acetate,the membrane permeability of Candida albicans cell was increased by modifying chemical treatment method,and further to change the efficiency of plasmid transformation. Methods：The recombinant plasmid was linearied using restriction enzyme,and then the transformatiom efficiency was detected through adding different concentrations of dimethyl suifoxide（DMSO）（1%,5%,and 10%）or taking various length of heat shock（0.5 h,2 h,3 h and 4 h）based on the traditional transformation methods. The optimum concentration of DMSO and the best length of heat shock were determined according to the transformation efficiency experiment. Finally,we cultured the Candida albicans on the plates of SC select medium using these two methods to compare the plasmid transformation efficiency between these two groups. Results：The efficiency of plasmid transformation using the common method is 0.6×10^5transformants/1 μg plasmid DNA/10^8 cells,while the efficiency of that using the improved method is 1.5×10^5transformants/1 μg plasmid DNA/108 cells.The differences between those two groups were statistically significant. Conclusion：The improved method increases the Candida albicans plasmid transformation efficiency.