Electroporation-mediated transformation of Sclerotium rolfsii

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.