Targeted Gene Deletion in Cordyceps militaris Using the Split-Marker Approach
The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.
KeywordsCordyceps militaris Homologous recombination Split-marker Filamentous fungus Protoplast
This work was supported by the National Natural Science Foundation of China (Grant Nos. 31572178, 31372116), and the Projects of Science and Technology of Guangdong Province (Grant Nos. 2014B020205003, 2016A030313404). We are grateful to Prof. Gang Liu and Yuanyuan Pan, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, for providing pAg1-H3 vector.
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