电穿孔法转化齐整小核菌

Electroporation-mediated transformation of Sclerotium rolfsii

摘要:

【目的】建立齐整小核菌(Sclerotium rolfsii)的电穿孔法转化方案，以实现经济、快速地遗传转化。【方法】将齐整小核菌gpd基因启动子控制的basta抗性基因bar与红色荧光蛋白基因DsRed Max组成的融合蛋白表达盒，通过电击转入野生型齐整小核菌细胞中，筛选转化子并进行PCR与荧光观察验证。在此基础上，测试了不同电压、脉冲时间、外源DNA片段与受体细胞比例等条件下的转化效率，以得到优化的电转化参数。最后，采用优化的条件，尝试转化多种抗性基因与荧光蛋白融合表达盒以测试其可用性。【结果】成功得到了bar、sdhR与aphI基因的转化子。【结论】成功建立了优化的齐整小核菌电穿孔转化法。优化的参数条件为电压2 kV/cm、脉冲时间1 ms、DNA/匀浆细胞比例3 μg/300 mg，电击1次。

Abstract:

[Objective] To establish an electroporation-mediated transformation method for the economical and rapid genetic transformation of Sclerotium rolfsii. [Methods] The fusion protein expression cassette composed of the basta-resistant gene bar and the red fluorescent protein gene DsRed Max, controlled by the promoter of the native gpd gene, was transferred into the wild-type cells of S. rolfsii. The transformants were screened and validated by PCR and fluorescence observation. Further, we tested the transformation efficiencies under the different conditions of field strength, pulse time, and the ratio of foreign DNA fragments to recipient cells, to figure out the optimized electroporation parameters. Finally, we transformed the expression cassettes fusing multiple resistance genes and DsRed under optimized conditions to test their availabilities. [Results] The transformants carrying gene bar, sdhR, and aphI were obtained successfully. [Conclusion] The electroporation-mediated transformation method of S. rolfsii was successfully established. The optimized transformation parameters were field strength of 2 kV/cm, pulse time of 1 ms, DNA/homogenized cell ratio of 3 μg/300 mg, and pulse once.