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.
Highlights
Ganoderma lucidum is one of the most widely cultivated pharmaceutical mushrooms in East Asia because it contains many pharmacologically active compounds, such as ganoderic acid and polysaccharide [1,2,3]
The 59 self-formed adaptor PCR (SEFA) and 39 rapid-amplification of cDNA ends (RACE) methods were utilized to obtain the fulllength sequence of the gene
Because of the uncertainty regarding the presence of a genesilencing mechanism in some fungi, such as Saccharomyces cerevisiae and Ustilago maydis [40], we identified the presence of a Dicer-1 homolog in the G. lucidum genome (Fig. S1, Fig. S2, Fig. S3)
Summary
Ganoderma lucidum is one of the most widely cultivated pharmaceutical mushrooms in East Asia because it contains many pharmacologically active compounds, such as ganoderic acid and polysaccharide [1,2,3]. Many studies have described the pharmacological activity and chemical composition of G. lucidum [4,5,6]. These pharmacological compounds, which inhibit tumor cell growth [7], modulate the immune system and lower blood sugar levels [8], are thought to be potential candidates for drug discovery, and the pharmacological value of G. lucidum has drawn widespread interest [9]. G. lucidum has been considered a genetically intractable organism, and tools for investigating the functional genomics of G. lucidum are still at an early stage, some gene transfer procedures have been established [22,23,24]. Improving the genetic tools for this organism will provide significant insight into the mechanism of synthesis of pharmacologically active compounds, open new possibilities for the exploitation of G. lucidum for enhancing pharmacologically active compounds by genetic engineering, and be critical for studying the biology, chemical composition, and evolution of this organism
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