ESTs analysis of putative genes engaged in Polyporus umbellatus sclerotial development

Abstract

Polyporus umbellatus is one of the most widely used and precious medicinal fungi and the underground sclerotia are known to be with great medicinal value. However, the molecular mechanisms involved in sclerotial development are poorly understood. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of Polyporus umbellatus to identify genes expressing differently between mycelium and sclerotia. In this library, a total of 1202 clones were sequenced, assembled into 222 contigs and 524 singletons which were further searched against the NCBI nonredundant (NR) protein database (E-value cutoff, 10-5). Based on sequence similarity with known proteins, 378 sequences between mycelium and sclerotial were identified and classified into different functional categories through Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs). We have finally identified a majority of differentially expressed genes (constituting 5.6% of the present library) between the two different periods. An expression level of 32 selected expressed sequence tags (ESTs) generated from the above SSH cDNA library was studied through RT-PCR. This study provides the first global overview of genes putatively involved in Polyporus umbellatus sclerotial development and provides a preliminary basis for further functional research in terms of regulated gene expression in sclerotial production.

Figure 1

Inducing sclerotial formation under artificial conditions. (a) Mycelia could not form sclerotia; (b) sclerotial formation after 60 days cultivation.

Figure 2

Representation of gene ontology assignments for fungal homologous expressed sequence tags (ESTs) derived from the suppression subtractive hybridization (SSH) library.

Figure 3

A graphical representation of sequence distribution based on Clusters of orthologous Groups of proteins (COGs).

Figure 4

Expression patterns of redox related genes from SSH library using qPCR analyses. Blue columns represent the Relative Quantification (RQ) in sclerotium; light grey columns indicate the RQ in mycelium. All standards were run in duplicate and samples were run in triplicate. Contig37: glyoxal oxidase; Contig83: choline oxidase; Contig110: NADPH oxidase; Contig75: glutathione peroxidase; 0004: peroxisomal membrane protein; 388: cysteine peroxidase; 551: thioredoxin-dependent peroxidase; 589: chloroperoxidase-like protein; 233: alcohol oxidase-like protein.

Figure 5

Expression patterns of signal related genes from SSH library using qPCR analyses. Blue columns represent the RQ in sclerotium; light grey columns indicate the RQ in mycelium. All standards are run in duplicate and samples are run in triplicate. Puctg838, Puctg616, Puctg1343, Pu13, Pu1168: small GTPase; Puctg20, Puctg13, Pu1317, Pu382, Pu488: protein kinase; Puctg126: G protein; Pu209: G protein-coupled receptor (GPCR).

Figure 6

Expression patterns of growth related genes from SSH library using qPCR analysis. Blue columns represent the RQ in sclerotium; light grey columns indicate the RQ in mycelium. All standards were run in duplicate and samples were run in triplicate. Pu1441: cytochrome P450; Puctg22, Puctg8: hemolysin; Pu892, Pu542: inhibitor of apoptosis; Pu191: Cyclins, Pu790: cyclophilin, Puctg202, Puctg112: hydrophobin; Pu1355: C-4 sterol methyl oxidase; Puctg88: hydrophobin.