Deep Insight into the Ganoderma lucidum by Comprehensive Analysis of Its Transcriptome

Background Ganoderma lucidum is a basidiomycete white rot fungus and is of medicinal importance in China, Japan and other countries in the Asiatic region. To date, much research has been performed in identifying the medicinal ingredients in Ganoderma lucidum. Despite its important therapeutic effects in disease, little is known about Ganoderma lucidum at the genomic level. In order to gain a molecular understanding of this fungus, we utilized Illumina high-throughput technology to sequence and analyze the transcriptome of Ganoderma lucidum. Methodology/Principal Findings We obtained 6,439,690 and 6,416,670 high-quality reads from the mycelium and fruiting body of Ganoderma lucidum, and these were assembled to form 18,892 and 27,408 unigenes, respectively. A similarity search was performed against the NCBI non-redundant nucleotide database and a customized database composed of five fungal genomes. 11,098 and 8, 775 unigenes were matched to the NCBI non-redundant nucleotide database and our customized database, respectively. All unigenes were subjected to annotation by Gene Ontology, Eukaryotic Orthologous Group terms and Kyoto Encyclopedia of Genes and Genomes. Differentially expressed genes from the Ganoderma lucidum mycelium and fruiting body stage were analyzed, resulting in the identification of 13 unigenes which are involved in the terpenoid backbone biosynthesis pathway. Quantitative real-time PCR was used to confirm the expression levels of these unigenes. Ganoderma lucidum was also studied for wood degrading activity and a total of 22 putative FOLymes (fungal oxidative lignin enzymes) and 120 CAZymes (carbohydrate-active enzymes) were predicted from our Ganoderma lucidum transcriptome. Conclusions Our study provides comprehensive gene expression information on Ganoderma lucidum at the transcriptional level, which will form the foundation for functional genomics studies in this fungus. The use of Illumina sequencing technology has made de novo transcriptome assembly and gene expression analysis possible in species that lack full genome information.

[1]  Carmen W Huie,et al.  A review of modern sample-preparation techniques for the extraction and analysis of medicinal plants , 2002, Analytical and bioanalytical chemistry.

[2]  Y. Hayashizaki.  Mouse Genome Encyclopedia Project. , 2003, Cold Spring Harbor symposia on quantitative biology.

[3]  Q. Zeng,et al.  Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus) , 2010, Proceedings of the National Academy of Sciences.

[4]  Kyle Bibby,et al.  Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta: Pathway description and gene discovery for production of next-generation biofuels , 2011, BMC Genomics.

[5]  Y. Van de Peer,et al.  The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis , 2008, Nature.

[6]  B. Henrissat,et al.  FOLy: an integrated database for the classification and functional annotation of fungal oxidoreductases potentially involved in the degradation of lignin and related aromatic compounds. , 2008, Fungal genetics and biology : FG & B.

[7]  A. Matuszewska,et al.  Biodegradation of lignin by white rot fungi. , 1999, Fungal genetics and biology : FG & B.

[8]  Vincent Lombard,et al.  Genome sequence of the model mushroom Schizophyllum commune , 2010, Nature Biotechnology.

[9]  Virginia Walbot,et al.  A green chapter in the book of life , 2000, Nature.

[10]  A. Weis,et al.  Therapeutic effects of substances occurring in higher Basidiomycetes mushrooms: a modern perspective. , 1999, Critical reviews in immunology.

[11]  Chengying Shi,et al.  Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds , 2011, BMC Genomics.

[12]  L. Ye,et al.  Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum. , 2001 .

[13]  Hai-Jun Xu,et al.  Transcriptome Analysis of the Brown Planthopper Nilaparvata lugens , 2010, PloS one.

[14]  Y. Kimura,et al.  Antitumor and antimetastatic effects on liver of triterpenoid fractions of Ganoderma lucidum: mechanism of action and isolation of an active substance. , 2002, Anticancer research.

[15]  Ya-Jie Tang,et al.  Role of oxygen supply in submerged fermentation of Ganoderma lucidum for production of Ganoderma polysaccharide and ganoderic acid , 2003 .

[16]  J. Yue,et al.  Sterols and triterpenoids from the spores of Ganoderma lucidum , 2008, Natural product research.

[17]  Inna Dubchak,et al.  The genome portal of the Department of Energy Joint Genome Institute: 2014 updates , 2013, Nucleic Acids Res..

[18]  Y. Benjamini,et al.  Controlling the false discovery rate in behavior genetics research , 2001, Behavioural Brain Research.

[19]  J. Watanabe,et al.  Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium. , 1989, The Journal of biological chemistry.

[20]  Ya-Jie Tang,et al.  A novel three‐stage light irradiation strategy in the submerged fermentation of medicinal Mushroom Ganoderma lucidum for the efficient production of ganoderic acid and Ganoderma polysaccharides , 2008, Biotechnology progress.

[21]  Mark Johnston,et al.  Benchmarking next-generation transcriptome sequencing for functional and evolutionary genomics. , 2009, Molecular biology and evolution.

[22]  Y. Li,et al.  Generation and analysis of expressed sequence tags from a cDNA library of the fruiting body of Ganoderma lucidum , 2010, Chinese medicine.

[23]  Ming Zhao,et al.  Cloning and sequence analysis of a glyceraldehyde-3-phosphate dehydrogenase gene from Ganoderma lucidum. , 2006, Journal of microbiology.

[24]  Q. Dong,et al.  Structure and Conformation Behavior of a Glucan from Spores of Ganoderma lucidum (Fr.) Karst. , 2000, Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica.

[25]  F. Feltus,et al.  The first genome-level transcriptome of the wood-degrading fungus Phanerochaete chrysosporium grown on red oak , 2009, Current Genetics.

[26]  Yong-Su Jin,et al.  Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis , 2007, Nature Biotechnology.

[27]  J. Watanabe,et al.  Immunomodulator, LZ-8, prevents antibody production in mice. , 1991, International journal of immunopharmacology.

[28]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[29]  Huanming Yang,et al.  De novo assembly of human genomes with massively parallel short read sequencing. , 2010, Genome research.

[30]  A. Oliphant,et al.  A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). , 2002, Science.

[31]  Mingwen Zhao,et al.  Cloning and Characterization of a Gene Encoding HMG-CoA Reductase from Ganoderma lucidum and Its Functional Identification in Yeast , 2008, Bioscience, biotechnology, and biochemistry.

[32]  Wei Zhao,et al.  Biotechnological production and application of ganoderic acids , 2010, Applied Microbiology and Biotechnology.

[33]  M. Rodríguez-Concepcíon,et al.  The MEP pathway: a new target for the development of herbicides, antibiotics and antimalarial drugs. , 2004, Current pharmaceutical design.

[34]  Luciano Milanesi,et al.  Data handling strategies for high throughput pyrosequencers , 2007, BMC Bioinformatics.

[35]  Jonathan D. G. Jones,et al.  Application of 'next-generation' sequencing technologies to microbial genetics , 2009, Nature Reviews Microbiology.

[36]  M. Prevost,et al.  Wood Degradation by White Rot Fungi: Cytochemical Studies Using Lignin Peroxidase-Immunoglobulin-Gold Complexes , 1987, Applied and environmental microbiology.

[37]  Chi‐Huey Wong,et al.  Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction , 2007, BMC Genomics.

[38]  Yizhou Yin,et al.  Ganodermataceae: natural products and their related pharmacological functions. , 2007, The American journal of Chinese medicine.

[39]  D. Sliva,et al.  Suppression of the inflammatory response by triterpenes isolated from the mushroom Ganoderma lucidum. , 2009, International immunopharmacology.

[40]  S. Joo,et al.  Molecular cloning and expression of a laccase from Ganoderma lucidum, and its antioxidative properties. , 2008, Molecules and cells.

[41]  Y. Leem,et al.  Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum , 2001, Applied Microbiology and Biotechnology.

[42]  J. Claverie,et al.  The significance of digital gene expression profiles. , 1997, Genome research.

[43]  E. Swiezewska,et al.  Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. , 2001, Acta biochimica Polonica.

[44]  S. Tanaka,et al.  Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (LZ-8). An immunomodulator from a fungus, Ganoderma lucidium, having similarity to immunoglobulin variable regions. , 1989, The Journal of biological chemistry.

[45]  K. Mizumoto,et al.  An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice , 1990, Diabetologia.

[46]  C. Hung,et al.  Antiinflammatory triterpenoids and steroids from Ganoderma lucidum and G. tsugae. , 2008, Phytochemistry.

[47]  Xiao-yu Li,et al.  Structural features of immunologically active polysaccharides from Ganoderma lucidum. , 2002, Phytochemistry.

[48]  Susan C. Jones,et al.  Transcriptomics of the Bed Bug (Cimex lectularius) , 2011, PloS one.

[49]  Shuqin Zhang,et al.  Recombinant Lz-8 from Ganoderma lucidum induces endoplasmic reticulum stress-mediated autophagic cell death in SGC-7901 human gastric cancer cells , 2011, Oncology reports.

[50]  T. Ng,et al.  A laccase from the medicinal mushroom Ganoderma lucidum , 2006, Applied Microbiology and Biotechnology.

[51]  C. Chien,et al.  Zebrafish genome project: bringing new biology to the vertebrate genome field. , 2007, Zebrafish.

[52]  O. Belova,et al.  Purification, crystallization and preliminary X-ray structure analysis of the laccase from Ganoderma lucidum. , 2011, Acta crystallographica. Section F, Structural biology and crystallization communications.

[53]  J. Zhong,et al.  Separation and determination of four ganoderic acids from dried fermentation mycelia powder of Ganoderma lucidum by capillary zone electrophoresis. , 2010, Journal of pharmaceutical and biomedical analysis.

[54]  Yi Ding,et al.  Molecular Cloning, Characterization, and Differential Expression of a Farnesyl-Diphosphate Synthase Gene from the Basidiomycetous Fungus Ganoderma lucidum , 2008, Bioscience, biotechnology, and biochemistry.

[55]  Peter E. Larsen,et al.  Using Deep RNA Sequencing for the Structural Annotation of the Laccaria Bicolor Mycorrhizal Transcriptome , 2010, PloS one.

[56]  P. Bisen,et al.  Ganoderma lucidum: a potent pharmacological macrofungus. , 2009, Current pharmaceutical biotechnology.

[57]  S. Salzberg,et al.  Bioinformatics challenges of new sequencing technology. , 2008, Trends in genetics : TIG.

[58]  Ya-Jie Tang,et al.  Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum. , 2009, Bioresource technology.

[59]  M. Huang,et al.  Mechanism of the antiulcerogenic effect of Ganoderma lucidum polysaccharides on indomethacin-induced lesions in the rat. , 2002, Life sciences.

[60]  Bernard Henrissat,et al.  Corrigendum: Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina) , 2008, Nature Biotechnology.

[61]  Toshitsugu Sato,et al.  Isolation and characterization of a fruiting body-specific exo-β-1,3-glucanase-encoding gene, exg1, from Lentinula edodes , 2005, Current Genetics.

[62]  Masaya Nakamura,et al.  Molecular cloning of developmentally specific genes by representational difference analysis during the fruiting body formation in the basidiomycete Lentinula edodes. , 2005, Fungal genetics and biology : FG & B.

[63]  Y. Lau,et al.  Ganoderma lucidum polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function , 2008, Journal of hematology & oncology.

[64]  Zhi-bin Lin,et al.  [Advances of pharmacological effects of triterpenes from Ganoderma lucidum]. , 2002, Yao xue xue bao = Acta pharmaceutica Sinica.

[65]  Lei Zhou,et al.  Characterization and immunostimulatory activity of a polysaccharide from the spores of Ganoderma lucidum. , 2009, International immunopharmacology.

[66]  Nan Wang,et al.  Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum. , 2007, Journal of microbiology and biotechnology.

[67]  X Bao,et al.  Structural and immunological studies of a major polysaccharide from spores of Ganoderma lucidum (Fr.) Karst. , 2001, Carbohydrate research.

[68]  Brian C Grieb,et al.  Ganoderic acids suppress growth and invasive behavior of breast cancer cells by modulating AP-1 and NF-kappaB signaling. , 2008, International journal of molecular medicine.

[69]  Shufeng Zhou,et al.  Antitumor activity and underlying mechanisms of ganopoly, the refined polysaccharides extracted from Ganoderma lucidum, in mice. , 2005, Immunological investigations.

[70]  Ya-Jie Tang,et al.  Improvement of ganoderic acid and Ganoderma polysaccharide biosynthesis by Ganoderma lucidum fermentation under the inducement of Cu2+ , 2009, Biotechnology progress.

[71]  R. Paterson,et al.  Ganoderma - a therapeutic fungal biofactory. , 2006, Phytochemistry.

[72]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[73]  Human genome: Genomes by the thousand , 2010, Nature.

[74]  Wenjia Yang,et al.  Transcriptome Analysis of the Oriental Fruit Fly (Bactrocera dorsalis) , 2011, PloS one.

[75]  Igor Grigoriev,et al.  Comparative Transcriptome and Secretome Analysis of Wood Decay Fungi Postia placenta and Phanerochaete chrysosporium , 2010, Applied and Environmental Microbiology.

[76]  L. Qin,et al.  Molecular Cloning, Characterization, and Differential Expression of a Lanosterol Synthase Gene from Ganoderma lucidum , 2010, Bioscience, biotechnology, and biochemistry.

[77]  Chuan-Xi Zhang,et al.  De novo characterization of a whitefly transcriptome and analysis of its gene expression during development , 2010, BMC Genomics.

[78]  A. Salamov,et al.  Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion , 2009, Proceedings of the National Academy of Sciences.

[79]  Bernard Henrissat,et al.  Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina) , 2008, Nature Biotechnology.

[80]  Cong-Zhao Zhou,et al.  Crystal structure of LZ‐8 from the medicinal fungus Ganoderma lucidium , 2009, Proteins.

[81]  Katherine H. Huang,et al.  Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78 , 2004, Nature Biotechnology.

[82]  E. Uberbacher,et al.  CAZymes Analysis Toolkit (CAT): web service for searching and analyzing carbohydrate-active enzymes in a newly sequenced organism using CAZy database. , 2010, Glycobiology.

[83]  A. Salamov,et al.  Computational analysis of the Phanerochaete chrysosporium v2.0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins. , 2006, Fungal genetics and biology : FG & B.

[84]  Huanming Yang,et al.  A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. indica) , 2002, Science.