title: A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.
abstract: The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. 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% (RdRP1), 85% (RdRP2) and 77% (RdRP3). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum.
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China; College of Agricultural Science and Technology, Chinese Agricultural University, Beijing 100081, China; College of Life Science, Shanxi Normal University, Linfen 041004, China.
authors.forename: Liqin
authors.initials: L
authors.lastname: Liang
authors.affiliation: College of Agricultural Science and Technology, Chinese Agricultural University, Beijing 100081, China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Life Science, Shanxi Normal University, Linfen 041004, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Cheng
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Ling
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Zhongqin
authors.initials: Z
authors.lastname: Luo
authors.affiliation: College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, China.
authors.forename: Miao
authors.initials: M
authors.lastname: Bai
authors.affiliation: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Electronic address: xieby@mail.caas.net.cn.
authors.forename: Bingyan
authors.initials: B
authors.lastname: Xie
mesh_terms: D015415:Biomarkers / Q000032:analysis*; D018274:Electroporation; D005656:Fungal Proteins / Q000235:genetics*; D005670:Fusarium / Q000235:genetics*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*
chemical_list: D015415:Biomarkers; D005656:Fungal Proteins
keywords: Electroporation; Fusarium oxysporum; Homologous Disruption; Recombination; Split-marker