Authors
Feng, X., Sheng, X., Liu, J., Zhou, R., Yang, Z., Tang, X., Zhang, S.
Abstract
Ophiocordyceps xuefengensis is a newly identified medicinal fungus with significant pharmacological and economic value, but its genetic manipulation has been impeded by the lack of an efficient transformation system. Here, we established the first stable and highly efficient polyethylene glycol (PEG)-mediated protoplast transformation platform for O. xuefengensis using Hygromycin B as a selectable marker. Through systematic optimization of critical parameters such as enzyme composition, enzyme concentration, mycelial age, and digestion conditions, we developed an optimized protocol for protoplast preparation. A high protoplast yield of 9.42x107CFU/mL was achieved using 4-day-old mycelia with 1.5% lywallzyme 1 and 1.5% snailase digested at 34{degrees}C with shaking at 130 rpm for 3.5 h. The PY medium containing 0.6 M mannitol significantly enhanced protoplast regeneration. Stable plasmid integration and robust hygR gene expression were confirmed through PCR detection and sustained antibiotic resistance over four successive generations. Furthermore, a controllable expression system was established by using the endogenous promoter that driven the stable expression of the glycoside hydrolase gene cbhI. The result of enzymatic assay confirmed the functional production of CBHI enzyme, demonstrating the credibility of this system. This work provides a reliable genetic toolbox for functional genomics studies, targeted gene manipulation, and strain engineering in O. xuefengensis, facilitating fundamental research and sustainable utilization of this valuable medicinal species.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 04 Nov 2025.
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