A high efficiency gene disruption strategy using a positive–negative split selection marker and electroporation for Fusarium oxysporum

In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10μF capacitance, 300Ω resistance, 4kV/cm field strength, with 1μg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per μg DNA. And after positive–negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP 1 ), 85% (RdRP 2 ) and 77% (RdRP 3 ). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum.
Source: Microbiological Research - Category: Infectious Diseases Source Type: research