Construction of Agrobacterium-mediated auxotrophic strain and genetic transformation system of Cordyceps militaris

Abstract:

[Background] Natural Cordyceps militaris is the fruiting body formed by Cordyceps militaris infects insect pupae or insects and has crucial biological and pharmacological activities. At the moment, the genome of C. militaris has been sequenced, but the research in molecular biology is limited. [Objective] To construct the Agrobacterium tumefaciens-mediated transformation (ATMT) system in C. militaris with uridine/uracil auxotrophic gene as the selection marker. [Methods] Orotidine-5'-monophosphate (OMP) decarboxylase is an essential enzyme for uracil synthesis. By using ATMT, We first knocked out the gene encoding pyrG of OMP decarboxylase in wild C. militaris by homologous recombination, and then constructed the uridine/uracil auxotrophic mutant. Afterward, the pyrG-carrying binary vector of Aspergillus oryzae was used to construct the complementary strain by ATMT. [Results] The pyrG was successfully knocked by homologous recombination and the uridine/uracil auxotrophic mutant was constructed. Under this background, the transformation efficiency reached (75±35)/10⁶ conidia after co-culture for 66 h at 22 ℃. In addition, the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter PgpdA and α-amylase promoter PamyB, which are commonly used by filamentous fungi, failed to drive the expression of GFP or DsRed reporter gene in C. militaris, but the promoter IF of C. militaris extension factor could well express GFP. [Conclusion] We constructed an ATMT system with uracil auxotrophic as a selection marker, providing a promising genetic tool for research on recombinant expression and gene function of C. militaris.