电穿孔介导马尔尼菲青霉转化体系的建立和优化

Electroporation-mediate transformation of Penicillium marneffei and optimization of the transforming conditions

目的建立和优化马尔尼菲青霉电穿孔转化体系,为其基因功能研究提供良好平台。方法直接使用马尔尼菲青霉缺陷株SPM4(pyrG-,niaD-)萌发孢子进行电穿孔转化,并通过改变孢子龄、孢子萌发时间、质粒浓度、电场强度等影响因素对体系进行优化。结果适合SPM4电穿孔转化条件为:孢子龄为6 d,孢子萌发时间为4 h,电场强度为5 kV/cm。上述条件下分别使用1μg环状或线性化质粒DNA转化SPM4,平均可得到21个和13个转化子。结论马尔尼菲青霉电穿孔转化效率高,重复性好,针对SPM4萌发孢子,环状质粒较线性化质粒电穿孔转化效率高。

Objective To establish and optimize the electroporation-mediated transformation system for Penicillium marneffei.It would provide a good platform for the gene function research of P.marneffei.Method The selectable marker pyrG gene was transformed into the germinated spores of Penicillium marneffei strain SPM4 （pyrG-,niaD-） by electroporation method.It aimed at finding out the optimal protocol by changing some important effecting factors including spore stadium,germination time,electric field intensity,and so on.Results The appropriate electroporation conditions for SPM4 were as follows：spore stadium 6 d,germination time 4 h and electric field intensity 5 kV/cm.Under these conditions,21 and 13 transformants were obtained using 1 μg circular and linearized plasmid DNA,separately.Conclusion Electroporation-mediate transformation of Penicillium marneffei can get high transformation efficiency and repeatability.It is implied that circular plasmid got higher transformation frequency than the linearized by electroporation for the germinated spores of SPM4.