Cloning of the ben gene and its functional identification in Cordyceps militaris

Selectable marker genes are very important for the screening of target mutants. At least two selectable marker genes are usually required to identify the function of a gene by gene knockout, gene complementation, and gene overexpression. However, there are few selectable marker genes that can be applied to Cordyceps militaris. In order to obtain the selectable marker gene which can be used to C. militaris, the sensitivity of C. militaris to benomyl as a fungicide was studied by the concentration gradient method. Then the benomyl resistance gene (the ben gene) was cloned from Neurospora crassa. The binary plasmid of the ben gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the ben gene in C. militaris was identified by the Agrobacterium tumefaciens-mediated transformation (ATMT) method. The results showed that 3 mu g/mL benomyl could completely inhibit the growth of C. militaris. The ben gene with a length of 1344 bp was successfully cloned from N. crassa. The binary plasmid pCAMBIA0390-Ben containing the ben gene expression cassette was efficiently constructed by the DJ-PCR method and the homologous recombination method. The ben gene of the plasmid pCAMBIA0390-Ben was successfully integrated into the genome of C. militaris by the ATMT method, and the C. militaris transformants with resistance to benomyl were obtained. Moreover, the ben gene could be highly expressed in C. militaris. It was confirmed that the ben gene derived from N. crassa could be used as a selectable marker gene in C. militaris. These results provide technical support for the identification of C. militaris gene function and the elucidation of the biosynthetic pathways of bioactive components in C. militaris.