Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Construction of a New Agrobacterium tumefaciens-Mediated Transformation System based on a Dual Auxotrophic Approach in Cordyceps militaris

  • Huan huan Yan (College of Life Sciences, Jiangxi Science and Technology Normal University) ;
  • Yi tong Shang (College of Life Sciences, Jiangxi Science and Technology Normal University) ;
  • Li hong Wang (College of Life Sciences, Jiangxi Science and Technology Normal University) ;
  • Xue qin Tian (College of Life Sciences, Jiangxi Science and Technology Normal University) ;
  • Van-Tuan Tran (VNU University of Science) ;
  • Li hua Yao (College of Life Sciences, Jiangxi Science and Technology Normal University) ;
  • Bin Zeng (Shenzhen Technology University) ;
  • Zhi hong Hu (College of Life Sciences, Jiangxi Science and Technology Normal University)
  • Received : 2023.12.06
  • Accepted : 2024.03.08
  • Published : 2024.05.28
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Abstract

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

Keywords

Acknowledgement

This work was supported by National Natural Science Foundation of China (NSFC Grant NO.32260009 and 31960193), and "Double Thousand Plan" in Jiangxi Province (jxsq2019201011).

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