The Development and Application of a Multiple Gene Co-Silencing System Using Endogenous URA3 as a Reporter Gene in Ganoderma lucidum
Open Access
- 24 August 2012
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 7 (8), e43737
- https://doi.org/10.1371/journal.pone.0043737
Abstract
Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5′-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.Keywords
This publication has 56 references indexed in Scilit:
- Efficient implementation of RNA interference in the pathogenic yeast Cryptococcus neoformansJournal of Microbiological Methods, 2011
- Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidumWorld Journal of Microbiology and Biotechnology, 2011
- Suppression of polygalacturonase gene expression in the phytopathogenic fungus Ophiostoma novo-ulmi by RNA interferenceFungal Genetics and Biology, 2010
- Establishing Molecular Tools for Genetic Manipulation of the Pleuromutilin-Producing FungusClitopilus passeckerianusApplied and Environmental Microbiology, 2009
- The NCBI BioSystems databaseNucleic Acids Research, 2009
- Self-Formed Adaptor PCR: a Simple and Efficient Method for Chromosome WalkingApplied and Environmental Microbiology, 2007
- An Efficient Fungal RNA-Silencing System Using the DsRed Reporter GeneApplied and Environmental Microbiology, 2007
- Pfam: clans, web tools and servicesNucleic Acids Research, 2006
- Mechanisms of gene silencing by double-stranded RNANature, 2004
- Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT MethodMethods, 2001