Construction and application of a gene silencing system using a dual promoter silencing vector in Hypsizygus marmoreus.

Abstract

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.