Cordyceps militaris, an ascomycete caterpillar fungus, has been used as a traditional Chinese medicine for many years owing to its anticancer and immunomodulatory activities. Currently, artificial culturing of this beneficial fungus has been widely used and can meet the market, but systematic molecular studies on the developmental stages of cultured C. militaris at transcriptional and translational levels have not been determined. We utilized high-throughput Illumina sequencing to obtain the transcriptomes of C. militaris mycelium and fruiting body. All clean reads were mapped to C. militaris genome and most of the reads showed perfect coverage. Alternative splicing and novel transcripts were predicted to enrich the database. Gene expression analysis revealed that 2,113 genes were up-regulated in mycelium and 599 in fruiting body. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to analyze the genes with expression differences. Moreover, the putative cordycepin metabolism difference between different developmental stages was studied. In addition, the proteome data of mycelium and fruiting body were obtained by one-dimensional gel electrophoresis (1-DGE) coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). 359 and 214 proteins were detected from mycelium and fruiting body respectively. GO, KEGG and Cluster of Orthologous Groups (COG) analysis were further conducted to better understand their difference. We analyzed the amounts of some noteworthy proteins in these two samples including lectin, superoxide dismutase, glycoside hydrolase and proteins involved in cordycepin metabolism, providing important information for further protein studies. The results reveal the difference in gene expression between the mycelium and fruiting body of artificially cultivated C. militaris by transcriptome and proteome analysis. Our study provides an effective resource for the further developmental and medicinal research of this promising fungus.

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. 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. 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.

Agrocybe aegerita, the black poplar mushroom, has been highly valued as a functional food for its medicinal and nutritional benefits. Several bioactive extracts from A. aegerita have been found to exhibit antitumor and antioxidant activities. However, limited genetic resources for A. aegerita have hindered exploration of this species. To facilitate the research on A. aegerita, we established a deep survey of the transcriptome and proteome of this mushroom. We applied high-throughput sequencing technology (Illumina) to sequence A. aegerita transcriptomes from mycelium and fruiting body. The raw clean reads were de novo assembled into a total of 36,134 expressed sequences tags (ESTs) with an average length of 663 bp. These ESTs were annotated and classified according to Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Gene expression profile analysis showed that 18,474 ESTs were differentially expressed, with 10,131 up-regulated in mycelium and 8,343 up-regulated in fruiting body. Putative genes involved in polysaccharide and steroid biosynthesis were identified from A. aegerita transcriptome, and these genes were differentially expressed at the two stages of A. aegerita. Based on one-dimensional gel electrophoresis (1-DGE) coupled with electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS), we identified a total of 309 non-redundant proteins. And many metabolic enzymes involved in glycolysis were identified in the protein database. This is the first study on transcriptome and proteome analyses of A. aegerita. The data in this study serve as a resource of A. aegerita transcripts and proteins, and offer clues to the applications of this mushroom in nutrition, pharmacy and industry.

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo.

Background: Non-small-cell lung cancer (NSCLC) is the main type of lung cancer with high mortality rates in worldwide. There is a need to identify better biomarkers to detect NSCLC at an early stage as this will improve therapeutic effect and patient survival rates. Methods: Two lectins (AAL/AAGL and AAL2/AANL), which specifically bind to tumour-related glycan antigens, were first used to enrich serum glycoproteins from the serum of early NSCLC patients, benign lung diseases subjects and healthy individuals. The samples were investigated by using iTRAQ labelling and LC-MS/MS. Results: A total of 53 differentially expressed proteins were identified by quantitative proteomics and four glycoproteins (AACT, AGP1, CFB and HPX) were selected for further verification by western blotting. Receiver operating characteristic analysis showed AACT was the best candidate for early NSCLC diagnosis of the four proteins, with 94.1% sensitivity in distinguishing early tumour Stage (IA+IB) from tumour-free samples (healthy and benign samples, HB). The GlcNAcylated AACT was further detected by lectin-based ELISA and has better advantage in clinical application than total AACT. The GlcNAcylated AACT can effectively differentiate Stage I from HB samples with an AUC of 0.908 and 90.9% sensitivity at a specificity of 86.2%. A combination of GlcNAcylated AACT and carcinoembryonic antigen (CEA) was able to effectively differing Stage I from HB samples (AUC=0.914), which significantly improve the specificity of CEA. The combination application also has the better clinical diagnostic efficacy in distinguishing cancer (NSCLC) from HB samples than CEA or GlcNAcylated AACT used alone, and yielded an AUC of 0.817 with 93.1% specificity. Conclusions: Our findings suggest that the GlcNAcylated AACT will be a promising clinical biomarker in diagnosis of early NSCLC.

The lectin from Agrocybe aegerita (AAL) has been found to possess potent tumor-suppressing function and tumor cell apoptosis-inducing activity. In this paper, we report the full sequence, the active expression of the gene encoding AAL at a high level and bioassay of the binding property with lactose, apoptosis-inducing activity and DNase activity of recombinant AAL (rAAL). The results reveal that AAL is a member of the galectin family and the dimeric form is the active unit for the functional performance. The rAAL showed comparable tumor cell apoptosis-inducing activity with the wild AAL but no DNase activity at all. The molecular characters revealed by this study are significant for the in-depth investigation of the functional mechanism of this interesting protein.