An efficient genetic transformation system for Chinese medicine fungus Tolypocladium ophioglossoides.

Tolypocladium ophioglossoides is a rare and valuable fungus extensively used in Chinese medicine for relieving postmenopausal syndrome in women yet its bioactive molecules are unknown. To explore its molecular mechanisms, we have developed a reliable Agrobacterium-mediated transformation system using the selective marker: the chlorimuron ethyl-resistance gene sur. For this purpose, we firstly constructed a T-DNA binary vector system and then improved the transformation efficiency by optimizing conditional parameters including the Agrobacterium tumefaciens concentration, the conidia number of T. ophioglossoides, the co-culture time and the concentration of acetosyringone. Furthermore, we have knocked-out the ku70 gene，which is a key gene in non-homologous end joining (NHEJ) DNA repair pathway，and the effect of the length of the homologous arms (HA) on the genetic transformation efficacy was also examined, which increased by 60% when HA was about 3 kb in length. Our results suggest that the genetic transformation system is efficient and feasible for the truffle-parasite fungus T. ophioglossoides, which can further be used in large-scale experiments for characterization of genes of interest in future work.