Development of an Expression Plasmid and its Use in Genetic Manipulation of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes)

Abstract

We report the construction of a plasmid, pJW-EXP, designed for the expression of homologous and heterologous genes in Ganoderma lucidum. pJW-EXP was generated from the plasmid pMD19-T by inserting the G. lucidum glyceraldehyde-3-phosphate dehydrogenase gene promoter, the G. lucidum iron-sulfur protein subunit of succinate dehydrogenase gene terminator and the homologous carboxin-resistance gene as selection marker. This expression plasmid can be efficiently transformed into Ganoderma through polyethylene glycol-mediated protoplast transformation. Southern blot analysis showed that most of the integrated DNA appeared as multiple copies in the genome. The applicability of the constructed plasmid was tested by expression of the truncated G. lucidum 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene that encodes the catalytic domain of HMGR. Overexpression of the truncated HMGR gene, which is a key gene in the biosynthetic pathway of the antitumor compounds, ganoderic acids, increased the transcription of the HMGR gene and enhanced ganoderic acid accumulation. pJW-EXP can serve as a useful tool in the genetic improvement and metabolic engineering of Ganoderma.

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system

“…The preparation of G. lucidum protoplasts and PEG‐mediated genetic transformation were conducted as previously described (Xu et al , , ; Yu et al , ). Transformants were screened using carboxin (2 mg l −1 ) or phosphinothricin (150 mg l −1 ) on a CYM regeneration plate.…”

Genetic engineering ofGanoderma lucidumfor the efficient production of ganoderic acids

“…Mitotic stability analysis revealed that the introduced DNA is stably integrated in the genome of transformants obtained through ATMT and PMT methods. 6,8,9 The reproducibility of the ATMT and PMT methods have also been confirmed through the overexpression of different G. lucidum homologous genes. [8][9][10][11][12] PMT, REMI and electroporation methods usually result in the ectopic integration of DNA into the genome of G. lucidum with one or more copy numbers.…”

“…We previously developed a selection marker gene cbx R on the basis of a host-derived gene rather than heterologous genes; we then used cbx R to establish a stable genetic transformation system for G. lucidum. [8][9] Molecular hybridization analysis revealed that cbx R is stably integrated in the genome of the transformants. Moreover, the established homologous transformation system has been successfully used to overexpress 3-hydroxy-3-methyL-glutaryl coenzyme A reductase gene, 9 squalene synthase gene, 10 a-phosphoglucomutase gene, 11 and UDP glucose pyrophosphorylase gene 12 in G. lucidum.…”

“…[8][9] Molecular hybridization analysis revealed that cbx R is stably integrated in the genome of the transformants. Moreover, the established homologous transformation system has been successfully used to overexpress 3-hydroxy-3-methyL-glutaryl coenzyme A reductase gene, 9 squalene synthase gene, 10 a-phosphoglucomutase gene, 11 and UDP glucose pyrophosphorylase gene 12 in G. lucidum. Our results showed that G. lucidum protoplasts were more efficiently transformed to confer carboxin resistance than hygromycin and phosphinothricin resistance; this finding indicates that homologous marker genes may be more efficiently used to establish genetic transformation systems than other genes.…”

“…Reliable genetic transformation methods should be established for the genetic engineering of G. lucidum. For instances, Agrobacterium tumefaciens-mediated transformation (ATMT), 6,8 polyethylene glycol-mediated transformation (PMT), 9 restriction enzyme-mediated DNA integration (REMI), 5 and electroporation transformation 7 have been developed for the genetic engineering of G. lucidum. The transformation efficiency through ATMT is 10-15 transformants per 10 7 protoplasts of G. lucidum, and this finding is comparable to that through REMI, PMT and electroporation methods, which yield a transformation efficiency of 4-20 transformants per mg DNA per 10 7 protoplasts.…”