Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin‐resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9‐mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5’‐monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde‐3‐phosphate dehydrogenase gene intron in the Cas9 plasmid is 14–18 in 107 protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA‐directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.
Background
Ganoderma lucidum, a well-known medicinal mushroom, has received wide attention as a promising cell factory for producing bioactive compounds. However, efficient expression of heterologous genes remains a major challenge in Ganoderma, hindering metabolic regulation research and molecular breeding of this species.
Results
We show that the presence of glyceraldehyde-3-phosphate dehydrogenase gene (gpd) intron 1 at the 5′ end of, the 3′ end of, or within the heterologous phosphinothricin-resistant gene (bar) is efficient for its expression in G. lucidum. The enhanced expression of bar is exhibited by the higher accumulation of mRNA and increased amounts of protein. Moreover, the insertion of the gpd intron 1 in the β-glucuronidase gene (gus) elevates its mRNA accumulation and enzyme activity, which facilitates the use of this reporter gene in Ganoderma.
Conclusions
This study has demonstrated the importance of the introduction of gpd intron 1 for the efficient expression of bar and gus in G. lucidum. The presence of the gpd intron 1 in heterologous genes increases levels of mRNA accumulation and protein expression in basidiomycete Ganoderma. The developed method may be utilized in upregulating the expression of other heterologous genes in Ganoderma.
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