Establishment of an Agrobacterium tumefaciens-mediated gene transfer approach for the medicinal fungus Cordyceps militaris G12

Abstract

Cordyceps militaris is a valuable edible and medicinal mushroom, which has been demonstrated to possess beneficial ingredients such as adenosine, cordycepin, pentostatin, polysaccharides, and carotenoids. Therefore, this fungus represents a promising cell factory for investigating the molecular mechanism of fruiting body formation and biosynthesis of bioactive metabolites. The development of efficient transformation systems in C. militaris plays an important role in the genetic manipulation of this fungus. In the present study, a commercial strain of C. militaris isolated in Vietnam has been identified on the basis of morphological characteristics and the ITS (internal transcribed spacer) sequence of ribosomal DNA. This strain was designated as G12 and employed for the genetic transformation. Assays for antifungal sensitivity of the C. militaris G12 strain towards three antifungal compounds including hygromycin, nourseothricin, and phleomycin revealed that this C. militaris strain was inhibited only by hygromycin at the concentration of 200 μg/ml. Under optimised conditions for the genetic transformation of the strain G12 using the hygromycin resistance marker and Agrobacterium tumefaciens-mediated transformation method, the transformation efficiency could reach about 800 transformants per 10⁶ spores. Further, the transfer of the DsRed and GFP reporter genes into the genome of C. militaris G12 has been demonstrated to be successful by PCR analyses with the gene-specific primers.