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.

Totopocladium ophioglossoides是一种稀有且有价值的真菌，广泛用于中药中以缓解女性绝经后综合症，但其生物活性分子尚不清楚。为了探索其分子机制，我们已经开发出了一种可靠的农杆菌介导的转化系统，使用了选择性标记：对嘧磺隆具有抗性的基因sur。为此，我们首先构建了一个T-DNA二元载体系统，然后通过优化条件参数（包括根癌土壤杆菌浓度，嗜盐球菌的分生孢子数，共培养时间和乙酰丁香酮浓度）来提高转化效率。此外，我们淘汰了ku70基因是非同源末端连接（NHEJ）DNA修复途径的关键基因，并且还研究了同源臂长度（HA）对遗传转化功效的影响，当HA升高时，其增加了60％长度约为3 kb。我们的结果表明，该遗传转化系统对于块菌-寄生菌T.ophioglossoides是有效且可行的，可进一步用于大规模实验中以表征未来工作中感兴趣的基因。