Protoplasting, regeneration and transformation of medicinal mushroom Ganoderma multipileum using succinate dehydrogenase mutation gene as a selection marker

Abstract

Ganoderma multipileum is a medicinal mushroom possessing potent medicinal secondary metabolites. However, in order to improve metabolic activity via molecular breeding, optimized protoplasting, regeneration and genetic transformation systems are mandatory but remain unexplored in G. multipileum. Thus, we sought to fill this gap by testing different parameters for protoplasting, regeneration, and genetic transformation based on the carboxin resistance marker gene. According to our results, the best viable protoplasting conditions were pH 6.0, mycelia age 4 days, enzyme concentration 7.5 mg/mL, 0.6 M sucrose osmoticum and 3 h of enzyme digestion. In a transformation study, a carboxin resistance gene cassette was constructed using PCR mutagenesis and restriction digestion of the native succinate dehydrogenase subunit B gene in G. multipileum. A PCR-restriction fragment length polymorphism strategy was also applied by PCR point mutagenesis to create an AgeI restriction site. The transformation system using carboxin resistant cassette proved amenable. The Sdh gene structure showed an intron structure similar to that of other Basidiomycetes and Ascomycetes. Alignment and analysis of the Sdh gene sequences in numerous fungi revealed conservation at a carboxin resistant point mutation site that could be applied to a wide range of fungal species. Further Sdh gene phylogenetic analysis revealed congruence with species phylogeny but with low bootstrap supporting values.

pks63787, a Polyketide Synthase Gene Responsible for the Biosynthesis of Benzenoids in the Medicinal Mushroom Antrodia cinnamomea

“…Among the four isotonic buffer systems (potassium chloride, mannitol, sucrose, magnesium sulfate) used in a previous study, sucrose generated the highest protoplast yield and was chosen for use in this study. 29 As shown in Table 1, approximately 6.0 × 10 7 protoplasts/mL were generated using 0.8 M sucrose. The optimal conditions included the use of 4-day-old germlings, a digestion enzyme concentration of 50 mg/mL, and digestion for 4 h. For the regeneration rate, the optimal conditions were the use of 5-dayold germlings, 1.0 M sucrose, and digestion with lysing enzyme at 50 mg/mL for 3 h (Table 1).…”

“…The effects of different parameters on the yield of protoplasts and the protoplast regeneration rate were evaluated, including the use of mycelium or germlings from arthrospores, growth age, the concentration of lysing enzymes, the duration of digestion with lysing enzymes, and the concentration of sucrose. Among the four isotonic buffer systems (potassium chloride, mannitol, sucrose, magnesium sulfate) used in a previous study, sucrose generated the highest protoplast yield and was chosen for use in this study . As shown in Table , approximately 6.0 × 10 7 protoplasts/mL were generated using 0.8 M sucrose.…”

Evaluation and Improvement of Protocols for Ganoderma boninense Protoplast Isolation and Regeneration

“…The second method was adopted by Chou & Tzean (2016) with slight modifications. The mycelia were rinsed with 0.6 M sucrose before being transferred into a 50 mL flask containing 15 mL of osmotic solution (0.6 M sucrose in 50 mM potassium phosphate buffer at pH 6, 2% lysing enzyme & 0.02% Driselase).…”

“…Various osmotic stabilizers were added to the regeneration media to protect the protoplast against the osmotic pressure during regeneration, which has a major influence on the protoplast's regeneration frequency. For this purpose, various published regeneration media that have been published by Li et al (2006), Priyatno (2009), Chou and Tzean (2016), Yu et al (2014), Govender et al (2016), Ab Wahab et al (2019) and Czapek-Dox-sucrose agar (CDSC) (Zhou et al, 2008) with modifications, were tested for the regeneration of the protoplasts in this study. The colonies generated from protoplasts under different nutrient sources and osmotic stabilizers were compared to each other.…”

Functional Genomics of Aspergillus oryzae: Strategies and Progress

“…established the transformation system by Bm selection for A. oryzae by increasing the susceptibility of A. oryzae to Bm [14]. Furthermore, pyrithiamine (PT) and phleomycin resistance genes have also been applied as dominant selection markers for the transformation of A. oryzae [15,16]. However, most transformation systems with drug resistance markers require expensive antibiotics.…”