The Development and Application of a Multiple Gene Co-Silencing System Using Endogenous URA3 as a Reporter Gene in Ganoderma lucidum

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.

Functional analysis of the role of glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal branching and the regulation of ganoderic acid biosynthesis in Ganoderma lucidum

“…The GPx sequence (GL25996-R1) was based on the G. lucidum genome sequence (Chen et al, 2012). RNAi silencing vector pAN7-dual-GPx, containing the hygromycin phosphotransferase (hph) encoding gene, was transferred into G. lucidum with electroporation and the transformants were selected on CYM medium containing 100 lg/mL hygromycin B (Mu et al, 2012). Ten randomly selected transformants were grown on CYM plates without hygromycin B and re-plated for 6 generations.…”

“…All samples were examined as three independent replicates. Experiments were carried out in a Realplex2 System (Eppendorf, Germany) with the following thermal cycling profile: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Relative quantification of gene expression was performed using the housekeeping gene 18S (Mu et al, 2012), and analysis was performed using the Eppendorf Mastercycler ep realplex 2.2 software. Evaluation of gene expression was carried out by calculating the difference between the Ct value of the gene analyzed and the Ct value of 18S rRNA, used as a normalizer.…”

“…To investigate the role of GPx in physiological processes, the RNAi silencing vector pAN7-dual was used to express the senseantisense structure of GPx (Mu et al, 2012). We constructed a GPx-silencing vector, pAN7-dual-GPxi, which carries the hygromycin B (Hyg) resistance gene as a selectable marker, and the GPx-silenced fragment is shown in Fig.…”

“…The lack of basic research on G. lucidum limits the further development and utilization of this organism. Luckily, the development of methodological platforms for genome sequencing, genetic transformation and transcriptional gene silencing has made it possible to conduct additional research on G. lucidum (Chen et al, 2012;Mu et al, 2012;Shi et al, 2012). Because of these studies, G. lucidum is now considered an adequate model system for the study of complicated growth patterns and the regulation of secondary metabolic pathways in higher basidiomycetes.…”

Heat Stress Modulates Mycelium Growth, Heat Shock Protein Expression, Ganoderic Acid Biosynthesis, and Hyphal Branching of Ganoderma lucidum via Cytosolic Ca 2+

“…However, the scarcity of basic biological studies has hindered the further development of G. lucidum and its commercial clinical use. Recently, the complete genome sequence (11), transgenic system (12), and reliable reverse genetic tools of G. lucidum were gradually developed (13), offering opportunities for further basic biological studies. Furthermore, as a higher basidiomycete with bioactive secondary metabolites, G. lucidum has become a potential model system for evaluating how environmental factors regulate the development and secondary metabolism of basidiomycetes.…”