Abstract
A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.
Highlights
Genetic transformation is a powerful tool to investigate the function of a gene of interest and to perform molecular breeding of new strains with desired properties in a specific organism
Stable and unstable transformation in C. subvermispora We tested several plasmids containing a heterologous hygromycin-resistant marker gene to transform C. subvermispora protoplasts using a conventional polyethylene glycol (PEG)/CaCl2 protocol developed for P. ostreatus (Honda et al 2000) and C. cinerea (Binninger et al 1987)
The regeneration rate of C. subvermispora protoplasts was less than 0.1%, while that of P. ostreatus was more than 2%
Summary
Genetic transformation is a powerful tool to investigate the function of a gene of interest and to perform molecular breeding of new strains with desired properties in a specific organism. In filamentous fungi, studies have mainly focused on the stable expression of genes integrated in the host chromosome, except for gene expression by extrachromosomal plasmids with an autonomously replicating DNA sequence, AMA1 (Gems et al 1991). A transformant is expected to maintain and express the introduced gene stably during mitotic and meiotic cell divisions; in most studies, a stable transformation is the goal in molecular breeding to produce a strain with a desired phenotype suitable for either basic research or industrial applications. Ectopic integrations occur at random sites of the host chromosome with different copy numbers, which makes it difficult to use stable transformants in an assay of gene expression control sequence in filamentous fungi. Gene targeting or genome editing may be used for assay of a gene expression control sequence, these technologies can be applicable in a limited number of model mushrooms and it is laborious and time-consuming to obtain strains with a series of mutations in the target region
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