Generation and analysis of expressed sequence tags from a cDNA library of the fruiting body of Ganoderma lucidum
© Luo et al; licensee BioMed Central Ltd. 2010
Received: 15 October 2009
Accepted: 16 March 2010
Published: 16 March 2010
Little genomic or trancriptomic information on Ganoderma lucidum (Lingzhi) is known. This study aims to discover the transcripts involved in secondary metabolite biosynthesis and developmental regulation of G. lucidum using an expressed sequence tag (EST) library.
A cDNA library was constructed from the G. lucidum fruiting body. Its high-quality ESTs were assembled into unique sequences with contigs and singletons. The unique sequences were annotated according to sequence similarities to genes or proteins available in public databases. The detection of simple sequence repeats (SSRs) was preformed by online analysis.
A total of 1,023 clones were randomly selected from the G. lucidum library and sequenced, yielding 879 high-quality ESTs. These ESTs showed similarities to a diverse range of genes. The sequences encoding squalene epoxidase (SE) and farnesyl-diphosphate synthase (FPS) were identified in this EST collection. Several candidate genes, such as hydrophobin, MOB2, profilin and PHO84 were detected for the first time in G. lucidum. Thirteen (13) potential SSR-motif microsatellite loci were also identified.
The present study demonstrates a successful application of EST analysis in the discovery of transcripts involved in the secondary metabolite biosynthesis and the developmental regulation of G. lucidum.
Ganoderma lucidum (Curtis: Fr.) P. Karst, Lingzhi in Chinese, which belongs to the Polyporaceae family, has been used in China as medicine for centuries to promote health and longevity [1, 2]. In other countries, its fruiting body is used to treat a variety of ailments, such as cancers, hypertension, diabetes, and hepatitis, apart from being a dietary supplement [2–4]. G. lucidum is an anti-tumour agent that acts via immune modulation or stimulating cytokine production [5–7]. The bioactive constituents of G. lucidum include more than 120 different triterpenes and polysaccharides, proteins and other compounds [2, 8].
Genes involved in the triterpenoids biosynthesis pathways in G. lucidum including squalene synthase (SQS), farnesyl-Diphosphate Synthase (GlFPS) and HMG-CoA reductase (Gl -HMGR) were isolated and characterized [9–11]. Joo et al. identified a laccase gene (GLLac1) from G. lucidum. However, little is known about the molecular biology of its fruiting body and its secondary metabolism. Identification of expressed genes, in particular the transcript profile, of the G. lucidum fruiting body would be a key to understanding its molecular biology.
Expressed sequence tag (EST) analysis allows rapid and large-scale identification of uniquely expressed genes [13, 14]. The EST analysis was used in transcriptome analysis of Lentinula edode, Aspergillus niger, Ustilago maydis and Neurosphora crassa. Sequencing information from ESTs may help discover genes in the biosynthesis of secondary metabolites . Loo et al. identified a gene involved in the ricinoleic acid biosynthetic pathway . Recently, genes encoding enzymes involved in the biosynthesis of ginsenoside, triterpene saponin and diterpenes were identified [21–23]. EST sequencing identified simple sequence repeats (SSRs) for genetic mapping .
Using the EST analysis, the present study annotated functional genes involved in the biosynthesis of secondary metabolites and the developmental regulation of the fruiting body of G. lucidum. Unique sequences very similar to squalene epoxydase (SE) and farnesyl-diphosphate synthase (FPS) in this EST collection were identified. We also discussed several candidate transcripts possibly associated with the cellular development of G. lucidum, such as hydrophobin, MOB2, profilin and PHO84. Moreover, identifying SSRs in the EST data is useful in marker-assisted breeding programs.
RNA extraction and cDNA library construction
The fruiting body of G. lucidum was obtained from the co-author Jin Lan, who has long been engaged in Ganoderma research in the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. She authenticated the G. lucidum using the morphological identification approach and referred to the Fungi Identification Manual . Fifty (50) days after growing on the basswood medium at 25-30°C in a shade shelter, approximately 0.5 g was harvested and frozen in liquid nitrogen immediately. The mRNA of thefruiting body was isolated and purified directly with a Dynabeads (R) mRNA DIRECT™ kit (Invitrogen, USA) according to the manufacturer's recommendations. The cDNA library was constructed from purified mRNA with a Creator™ SMART™ cDNA Library Construction kit (Clontech, USA). The double-stranded cDNA was directionally ligated into the Sfi I restriction site of the pDNR-lib vector (Clontech, USA) and electroporated into a DH5α Escherichia coli strain (TakaRa, Japan).
EST sequencing, assembly and annotation
A total of 1,023 randomly selected clones were cultured in liquid LB medium containing 34 mg/l chloramphenicol and incubated overnight at 220 rpm in rotation and 37°C. Plasmid DNA was prepared with an Axyprep-96 plasmid kit (Axygen, USA). The plasmid DNA was submitted for direct sequencing from the 5' end with an M13 forward primer on an ABI 3730 DNA sequencer using BigDye 3.1 sequencing chemistry (Applied Biosystems, USA).
The ABI-formatted chromatogram sequences were processed automatically with a local EST analysis pipeline. The Phred/Phrap program was applied for trace files conversion and for base calling with quality assessment [26, 27]. The vector and low-quality regions were removed from the sequence with the Cross Match typically included in the Phred/Phrap program. The short sequences (less than 100 bp) and poly A/T tails were filtered from the EST database. The high quality ESTs were assembled into contigs (clusters of assembled ESTs) and singletons (sequences found only once) by Phrap .
The unique sequences were searched against public databases including the SwissProt , NCBI non-redundant protein (Nr)  and non-redundant nucleotide (Nt)  databases using BLAST  algorithm, with a E-value cut-off at 10-5. The functional categories of these unique sequences were classified by a broad category, including metabolism, energy production, cell signalling, cell defence and stress response, cell structure and growth, transcription, protein synthesis, protein degradation, transport and secretion as well as unclassified and unknown function.
The detection of simple sequence repeats (SSRs) from the total high-quality ESTs of the fruiting body of G. lucidum was performed with the Simple Sequence Repeat Identification Tool (SSRIT) . The SSRIT accepts FASTA-formatted sequence files and reports the sequence ID, SSR motif, number of repeats (di- and tri-nucleotide repeat units), repeat length and position of the SSR and the total length of the sequence in which the SSRs were found . The search parameters for the maximum motif-length group were set to hexamer and those for the minimum number of repeats were set to five.
Result and discussion
General characteristics of G. lucidum fruiting body cDNA library and ESTs
Overview of the characteristics of the cDNA library of G. lucidum fruiting body
Total number of clones sequenced
Total high quality ESTs
Total unique genes
Average length per unique sequence (bp)
Number of contigs
Number of singletons
Number of annotated unique sequences
Number of non-annotated unique sequences
Expressed profile of the unique sequences
Occurrence of ESTs in unique sequences
Number of ESTs in a unique sequence
Number of unique sequences
Highly expressed transcripts in G. lucidum fruiting body cDNA library
Hypothetical protein [Rattus norvegicus]
Cell wall-associated hydrolase [Capnocytophaga sputigena Capno]
Predicted protein [Coprinopsis cinerea okayama7#130]
Protein TAR1 [Kluyveromyces lactis]
Hydrophobin 2 [Lentinula edodes]
Elongation factor 1-alpha [Schizophyllum commune]
Predicted protein [Laccaria bicolor S238N-H82]
Predicted protein [Laccaria bicolor S238N-H82]
Acyl-CoA-binding protein [Chaetophractus villosus]
Elongation factor 2 [Debaryomyces hansenii]
40S ribosomal protein S11 [Schizosaccharomyces pombe]
Annotation of expressed sequence tags
The list of the annotated ESTs found in the fruiting body of G. lucidum is shown in Additional file 1. Sixty-two (62) ESTs showed sequence similarities to uncharacterized genes encoding hypothetical proteins that were omitted from the list. The unique sequences from this cDNA library were analyzed for similarities by performing BLAST searches against public databases, including SwissProt , Nr  and Nt . A total of 139 (23.2%) and 67 (11.2%) unique sequences were assigned a putative identity based on significant sequence similarities to at least one sequence in the Nr and Nt databases, respectively. These annotated unique sequences provide an available resource for application and basic microbiology. Furthermore, among the 879 ESTs only three (0.3%) ESTs were identified as homologues of previously reported nucleotides from G. lucidum in the GenBank database, indicating that the vast majority of the ESTs in our dataset were unique and new. The three unique sequences showed similarities to cytochrome c oxidase subunit 2 (GO447869), glyceraldehyde-3-phosphate dehydrogenase (GO447698) and FPS (GO447502) (Additional file 1).
Functional distribution of ESTs
The di- and tri-nucleotide repeats in G.lucidum fruiting body ESTs
Candidate genes involved in the biosynthesis of triterpenoids
EST analysis is an important tool to identify secondary metabolite genes in the fruiting body of G. lucidum. Triterpenoids, the major bioactive compounds in G. lucidum, are synthesized from acetyl-CoA in the isoprenoid pathway. While genes involved in the triterpenoid biosynthetic pathway including SQS, GlFPS and Gl-HMGR were cloned from and identified in G. lucidum[9–11], other genes for the key enzymes in this pathway are to be identified.
According to the studies of the triterpene biosynthesis [10, 36], SE and FPS are rate-limiting enzymes in catalyzing triterpenoid biosynthesis in G. lucidum. The unique sequence (GO447913) with 71% identity (E-value = 1.00-12) to SE and the unique sequence (GO447502) with 98% identity to FPS (E-value = 4.00-27) involved in triterpenoid biosynthesis were presented in our EST data. SE acts as an important regulatory enzyme in the triterpenoid biosynthetic pathway. SE (EC 22.214.171.124), a monooxygenase, converts squalene into 2,3-oxidosqualene [36, 37]. The enzyme requires molecular oxygen, flavin adenine dinucleotide (FAD), either NADH or NADPH depending on the organisms . Since the gene encoding SE has not been identified in G. lucidum, the information of the unique sequence (GO447913) will help identify and characterize the SE in G. lucidum. The EST for FPS (GO447502) shows sequence similarity to the GlFPS, suggesting that this EST is the partial sequence of the full-length GlFPS.
The genes encoding key enzymes involved in the triterpenoid biosynthesis, such as SQS, Gl-HMGR and others, are not present in this EST dataset, indicates a low abundance of these genes in the fruiting body of G. lucidum or incomplete sequencing of the library. The absence of ESTs associated with the polysaccharide biosynthesis, which should be abundant in G. lucidum, in this study may be due to the limited sequencing scale.
Candidate genes involved in regulation of G. lucidum development
Several transcripts present in the EST dataset encode the proteins that may be associated with the development processes of the fruiting body of G. lucidum. The unique sequence (GO447641) showed sequence similarity to the inorganic phosphate transporter PHO84 gene which controls the absorption of phosphate nutrition and regulates the development of Saccharomyces cerevisiae. MOB2 is a nonessential yeast gene and plays a role in the maintenance of ploidy . The unique sequences homologous to MOB2 (GO447972) and PHO84 (GO447641) may have the same functions as those in yeast. Hydrophobin is expressed specially in filamentous fungi and is important during the morphogenesis of fungi and the fruiting body development of mushrooms . The unique sequence homologous to hydrophobin 2 of Lentinula edodes in this cDNA library consisted of five ESTs (GO447695, GO447166, GO447364, GO447414, GO447512), suggesting its abundance in the fruiting body of G. lucidum. Suizu et al. (2008) reported that the ESTs for hydrophobins were also most frequently identified in the cDNA library of Lentinula edodes. Profilin is a universal small eukaryotic protein that binds to monomeric actin (G-actin) and is involved in diverse functions such as maintenance of cell structural integrity, cell mobility and growth factor signal transduction . The sequences (GO447955, GO447282) encoding profilin were present in the G. lucidum cDNA library. The important unique sequence encoding an argonaute-like protein (GO447302) may be involved in the RNAi pathway, suggesting a potential gene knock-out by RNA interference in G. lucidum. Cloning and characterization of these candidate genes is under way.
Limitations of the study
The ESTs sequenced in this study from the fruiting body of G. lucidum were insufficient to cover all functional genes, although this EST dataset showed some characteristics of gene expression in the fruiting body of G. lucidum.
The present study used EST analysis and identified the transcripts in the biosynthesis of secondary metabolites and the developmental regulation of G. lucidum. For example, the candidate transcript encoding SE, the rate-limiting enzyme in the triterpenoid biosynthesis, was identified. Several genes associated with the development processes of G. lucidum, such as hydrophobin, MOB2, profilin and PHO84, were also identified.
Basic Local Alignment Search Tool
expressed sequence tag
National Center for Biotechnology Information
NCBI non-redundant protein
NCBI non-redundant nucleotide
simple sequence repeats
This study was supported by the National Natural Science Foundation of China (30772735) and the National Science & Technology Pillar Program in the 11th Five-year Plan of China (2006BAI09B02-1). We thank Dr. Haibo Sun from MininGene Biotechnology (Beijing, China) for his kind help in EST analysis.
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