Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 176, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.mimet.2020.106032
Keywords
Traditional Chinese fungi; Tolypocladium ophioglossoides; Agrobacterium tumefaciens; Genetic manipulation; Agrobacterium-mediated T-DNA transformation
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Funding
- Science and Technology Planning Project of Zhejiang Province, China [2017C33137]
- National Natural Science Foundation of China [31370064]
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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 trufle-parasite fungus T. ophioglossoides, which can further be used in large-scale experiments for characterization of genes of interest in future work.
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