Abstract
Fusarium graminearum is a destructive phytopathogen and shows an impressive metabolic diversity. Gene deletion is an important and useful approach for gene function study. Here we present a protocol for generating gene deletion mutant by applying “split-marker” deletion strategy (Catlett et al., 2003) with PEG-mediated protoplast transformation (Yuan et al., 2008; Martín, 2015).
Keywords: Fusarium graminearum, Gene knock out, Split-marker recombination
Materials and Reagents
Equipment
Procedure
For generating single gene deletion mutants, a variety of genes conferring resistance to antibiotics are available, e.g., genes resistant to Hygromycin B, Geneticin/G418, Bialaphos/Phosphothricin, Nourseothricin, Blasticidin, and Phleomycin. Among these, the gene resistant to Hygromycin B is the most widely used. In this protocol, we use hph as the resistant gene. Other markers can also be chosen according to your experiment as well (Turgeon et al., 2010).
Notes
Recipes
Acknowledgments
This protocol was adapted from previous work (Catlett et al., 2003; Zhang et al., 2012). This work was supported by the Natural Science Foundation of China (Grant 31730077) and the Ministry of Agriculture of China (Grant 2016ZX08009-003). The authors declare that there is no conflict of interest.
References
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