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As efficient reverse genetic tools are lacking, molecular genetics research has been limited in Hypsizygus marmoreus. In this study, we firstly constructed a gene-silencing method using a dual promoter vector (DPV) which was driven by gpd and 35 S promoters. The DPV was introduced into H. marmoreus via a simple electroporation procedure and the highest silenced rate of ura3 gene was 76.6%, indicating that the DPV might be suitable for gene silencing in basidiomycete. In this silencing system, the endogenous orotidine 5′-monophosphate decarboxylase gene ( ura3) was used as a selectable marker. Besides, we also constructed another silencing system which could silence the ura3 and other genes ( lcc1 encoded laccase1) together in H. marmoreus, and named it as co-silencing system. In the co-silenced transformants, we found that the mycelia were thinner and the growth was slower than in the wild-type and control2 strains, which was accordant with the previous study of lcc1 gene, indicating that the selective efficiency of the RNAi-mediated silencing of several genes might be increased by co-silencing ura3. The development of this molecular tool might improve functional studies of multiple genes in the basidiomycete H. marmoreus and also provide a reference for studies of other basidiomycetes. [ABSTRACT FROM AUTHOR]

The Hypsizygus marmoreus laccase gene ( lcc1 ) sequence was cloned and analyzed. The genomic DNA of lcc1 is 2336 bp, comprising 13 introns and 14 exons. The 1626-bp full-length cDNA encodes a mature laccase protein containing 542 amino acids, with a 21-amino acid signal peptide. Phylogenetic analysis showed that the lcc1 amino acid sequence is homologous to basidiomycete laccases and shares the highest similarity with Flammulina velutipes laccase. A 2021-bp promoter sequence containing a TATA box, CAAT box, and several putative cis-acting elements was also identified. To study the function of lcc1 , we first overexpressed lcc1 in H. marmoreus and found that the transgenic fungus producing recombinant laccase displayed faster mycelial growth than the wild-type (wt) strain. Additionally, primordium initiation was induced 3–5 days earlier in the transgenic fungus, and fruiting body maturation was also promoted approximately five days earlier than in the wt strain. Furthermore, we detected that lcc1 was sustainably overexpressed and that laccase activity was also higher in the transgenic strains compared with the wt strain during development in H. marmoreus . These results indicate that the H. marmoreus lcc1 gene is involved in mycelial growth and fruiting body initiation by increasing laccase activity. [ABSTRACT FROM AUTHOR]

Hypsizygus marmoreus is an important industrialized mushroom, yet the lack of basic research on this fungus has hindered further development of its economic value. In this study, mycelia injured by scratching were treated with hydrogen-rich water (HRW) to investigate the involvement of the redox system in fruiting body development. Compared to the control group, damaged mycelia treated with HRW regenerated earlier and showed significantly enhanced fruiting body production. Antioxidant capacity increased significantly in damaged mycelia after HRW treatment, as indicated by higher antioxidant enzyme activities and antioxidant contents; the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were also reduced at the mycelial regeneration stage after treatment with HRW. Furthermore, genes involved in ROS, Ca 2+ , MAPK and oxylipin signaling pathways were up-regulated by HRW treatment. In addition, laccase and manganese peroxidase activities and mycelial biomass were higher after HRW treatment, suggesting that HRW might enhance the substrate-degradation rate to provide more carbon sources for fruiting body production. [ABSTRACT FROM AUTHOR]