[Establishment and optimization of Agrobacterium tumefaciens-mediated genetic transformation system for Polyporus umbellatus].

The unstable quality of Polyporus umbellatus sclerotia during cultivation is the key factor affecting the quality and yield of P. umbellatus sclerotia. In order to provide technical support for obtaining superior P. umbellatus by molecular breeding, the genetic transformation system mediated by Agrobacterium tumefaciens was studied in this paper. A. tumefaciens-mediated method was used to investigate the effects of antibiotic concentration, strain type, A. tumefaciens concentration, receptor material, infection time, co-culture time, and screening conditions on the genetic transformation efficiency of P. umbellatus. The transformants were screened and detected by hygromycin resistance marker genes, polymerase chain reaction(PCR) of specific primers, and fluorescence detection methods. The results showed that the A. tumefaciens GV3101 strain could genetically transfer P. umbellatus mycelium cells, and the optimal conditions for infection were as follows: the A. tumefaciens concentration A_(600 nm)= 0.6, P. umbellatus mycelium cells as receptor material, infection time of 30 min, and co-culture time of 3 days. The two-step screening method involving hygromycin of 9 and 13 µg·mL~(-1 )was the best screening condition. The results of hygromycin resistance screening, PCR detection of specific primers, and fluorescence detection showed that the exogenous gene eGFP had been transferred into the P. umbellatus mycelium cells, integrated into the genome, and successfully expressed. Under optimal conditions, the conversion efficiency could be increased to 2.3%, and the genetic transformation period was shortened from more than 90 days to less than 60 days. This study established and optimized the genetic transformation system of P. umbellatus mycelium cells mediated by A. tumefaciens, laying a foundation for the analysis of the molecular mechanism of P. umbellatus during growth and molecular breeding.

[A landscape of transcriptome analysis of three sclerotia growth stages in Polyporus umbellatus].

Polyporus umbellatus,a traditional Chinese precious medicine as long been used for eliminating dampness,diuresis and have effect on cancer,getting more and more popularly in China recently. And the developmental metabolic process of the medicinal fungus,P. umbellatus,has been gotten more attention. This study is for the first time to explore the three sclerotial growth stages in P. umbellatus,named " white Polyporus"( initial phase), " grey Polyporus"( developmental phase) and " black Polyporus"( mature phase),by utilizing the de novo transcriptome assembly analysis technology. Finally,we obtained 88. 12 Gb sequence containing85 235 unigenes( ≥200 bp) assembled and 100% were annotated. We identified genes differentially expressed among the three stages of the sclerotia and screened out MFSgst,ERG4/ERG24,WD40,Rho A,CYP450,PKS,GSase and CHS1,which may contribute to the production of medicinal secondary metabolites and the defense mechanism against the environmental stress and biological invasion. We did the qRT-PCR trial to verify our results,which is in line with expectations. Our results are purposed to unearth the molecular mechanism of the accumulation of active constituents in different stages of Polyporus sclerotia which can be applied in the production and protection of Polyporus effectively.

[Molecular cloning and prokaryotic expression of a type II ribosome inactivating protein from Polyporus umbellatus].

A type Ⅱ ribosome inactivating protein (RIP) gene was cloned from Polyporus umbellatus sclerotia by RT-PCR method. The full open reading frame cDNA sequence of this gene was 873 bp in length and encoded a 290-aa protein with a molecular weight of 32.33 kDa and an isoelectric point of 5.58. Multiple sequence alignment revealed that the deduced amino acids possessed conserved domains of RICIN superfamily protein. A neighbor joining phylogenetic analysis suggests that PuRIP was closely related to RIP in Marasmius oreades. Real time PCR results showed that this gene expressed in all tested tissues of P. umbellatus. Meanwhile, the expression of this gene was significantly up-regulated in the part infected by Armillaria mellea. This result suggested that this PuRIP might played important role with potential biotic stress tolerance of P. umbellatus. Otherwise, we successfully constructed the pET15b-PuRIP plasmid, produced and purified the His-PuRIP fusion protein, which would provide the basic material for polyclonal antibody preparation and gene function research.

[Molecular cloning and characterization of two kind of heat-shock protein gene from Polyporus umbellatus].

With RT-PCR approaches, the full-length cDNA of two heat shock protein genes were cloned from total RNA of the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of the Hsp90 was 2 091 bp, encoding 696 amino acid residues with a predicted molecular mass of 78.9 kDa. The full open reading frame cDNA sequence of the Hsp70 was 1 944 bp, encoding 647 amino acid residues with a predicted molecular mass of 70.5 kDa. The Hsp90 and Hsp70 protein contained the conservative structure domain, respectively. Phylogenetic analysis showed that Hsp90 and Hsp90 from Trametes versicolor were clustered into one group, Hsp70 and Hsp70 from Fistulina hepatica were clustered into one group. Real-time PCR analysis showed that, the expression of Hsp90 and Hsp70 in the infected part by Amillariella mellea was upregulated. The expression profiling of Hsp90 and Hsp70 showed same patterns underbiotic stress. The results indicate that these two genes may play an important role in response to Amillariella mellea infection.

Diversity Analysis of Polyporus umbellatus in China Using Inter-simple Sequence Repeat (ISSR) Markers.

Polyporus (P.) umbellatus, an endangered medicinal fungus in China, is distributed throughout most areas of the country. Thirty-seven natural P. umbellatus samples collected from 12 provinces in China were subjected to the inter-simple sequence repeat (ISSR) assay to investigate the genetic diversity within and among the 11 natural populations. Nine ISSR primers selected from 100 primers produced 88 discernible DNA bands, with 46 being polymorphic. The frequency of polymorphism varied from 19.57 to 93.48% with an average of 61.26% across all populations. At the population level, the within-population variance was much greater (92.04%) than the between-population variance (7.96%) as revealed by analysis of molecular variance. Eleven P. umbellatus populations were grouped into two major clusters, and the clustering pattern displayed four groups using the unweighted pair-group method with an arithmetic mean dendrogram. Principal coordinate analysis further indicated that the genetic diversity of P. umbellatus strains was unevenly distributed and displayed a clustered distribution pattern instead. Within these clusters, subgrouping (Henan and Hubei) and cluster II (Jilin and Heilongjiang) related to the geographic distribution were evident. The present study provides the first global overview of P. umbellatus diversity analysis in China, which may open up new opportunities in comparative genetic research on this medicinal fungus in other countries.

[Molecular cloning and characterization of four small GTPase genes from medicinal fungus Polyporus umbellatus].

Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.

[Molecular cloning and characterization of two oxidative stress related genes from medicinal fungus Polyporus umbellatus].

OBJECTIVE: To clone the NADPH gene (PuNOX) and Glyoxal oxidase gene (PuGLOX) from a medicinal fungus Polyporus umbellatus, and to carry out the bioinformatic analysis. METHODS: We used the Rapid Amplification of cDNA ends (RACE) technique to obtain the full length cDNA of these two genes. We used a series of bioinformatic tools to characterize physiochemical properties of the two deduced protein. The analyses of multiple alignment and phylogenetic trees were performed using Bioeditor and MEGA 5.0 softwares. RESULTS: The entire cDNA of PuNOX and PuGLOX were 1674 bp, 1723 bp in length and encoded a 557-amino acid protein and 515-amino acid protein with a molecular weight of 63.845 kDa and 55.891 kDa and the isoelectric point of 5.58 and 4.82, respectively. PuNOX had high identities (74 to 80%) with NADPH peroxidase from other fungus. From the evolutionary tree, PuNOX was closely related to that of Pleurotus ostreatus. PuGLOX had high identities (> 50%) with Glyoxal oxidases from various fungus. Phylogenetic tree analysis suggested that PuGLOX was closely related to that of Phanerochaete chrysosporium. CONCLUSION: Molecular characterization of the two oxidative stress related genes will be useful for further functional determination of the genes involved in the sclerotium development of Polyporus umbellatus.

Identification of differentially expressed genes involved in laccase production in tropical white-rot fungus Polyporus sp. PG15.

Carbon sources and copper ion are the main influencing factors on the production of fungal laccase. To investigate the regulation of carbon source and copper ion in laccase production on the molecular level in tropical white-rot fungus PG15, a comparative analysis of gene expression patterns was performed by cDNA-amplified fragment length polymorphism (AFLP) technique. Selective amplifications with 120 primer combinations allowed the identification of 92 differentially expressed transcript-derived fragments (TDFs), ranging from 200 to 750 bp in size. The TDFs were from PG15 supplemented with different carbon sources and copper ion concentrations, majority of which downregulated laccase production. Twenty-one fragments that matched the database were functionally annotated and analyzed according to the up- and downregulation patterns identified by cDNA-AFLP. These fragments were probably involved in laccase production at the metabolism, signal transduction, transcription, or post-translation levels. This study provides the first catalog of genes involved in laccase production, together with their putatively functional annotations. These data provide potential candidates for improving laccase production in fungi by marker-assisted selection or genetic engineering.

ESTs analysis of putative genes engaged in Polyporus umbellatus sclerotial development.

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.

Nox gene expression and cytochemical localization of hydrogen peroxide in Polyporus umbellatus sclerotial formation.

The effect of temperature shift on Polyporus umbellatus sclerotial development was investigated. Micromorphology of the sclerotia was observed by using scanning electron microscopy (SEM). The cytochemical localization of H2O2 expressed as CeCl3 deposition at the subcellular level was observed by using transmission electron microscopy (TEM). Nox gene expression in sclerotia and mycelia was detected by quantitative real-time PCR (qRT-PCR) analysis. In addition, superoxide dismutase (SOD) and catalase (CAT) specific activities increased during sclerotial development and decreased after the antioxidant diphenyleneiodonium (DPI) was used. Results indicated that the temperature shift treatment induced P. umbellatus sclerotial formation. Compared with the mycelia, the Nox gene was respectively upregulated by 10.577-, 30.984- and 25.469-fold in the sclerotia of SI, SD and SM stages respectively. During the sclerotial formation, H2O2 accumulation was observed in the cell walls or around the organelle membranes of the mycelial cells. The antioxidant DPI decreased the generation of H2O2 in mycelial cells. The specific activity of SOD and CAT levels was decreased significantly by DPI. The activity of the two antioxidant enzymes in the mycelia increased much more during sclerotial formation (p < 0.05). Oxidative stress was closely associated with sclerotial development in P. umbellatus induced by temperature shift treatment.

Genetic diversity of endangered Polyporus umbellatus from China assessed using a sequence-related amplified polymorphism technique.

Polyporus umbellatus (Pers.) Fries is an endangered medicinal fungus in China with in vivo anticancer activity, but its genetic information is lacking. Eight natural P. umbellatus strains collected from 7 provinces in China were subjected to sequence-related amplified polymorphism markers to estimate the level and pattern of genetic diversity. Forty-nine primer combinations generated 1219 highly reproducible and discernible loci, among which 1023 were polymorphic. The percentage of polymorphism varied from 35.71 to 96.30 with an average of 83.92. Genetic identity among all strains ranged from 0.15 to 0.78 with an average of 0.46. The unweighted pair group method with arithmetic mean dendrogram clustered 8 strains into 3 clusters, and the clustering pattern showed 3 groups. Principal coordinate analysis further indicated that the genetic diversity of P. umbellatus strains was unevenly distributed and instead displayed a clustered distribution pattern. A relatively high level of genetic diversity was maintained in 8 natural P. umbellatus strains, but its abundance might be subjected to environmental heterogeneity, and the population structure of co-evolved Armillaria species may be selected by nature under the specific microenvironment.

Molecular cloning and characterization of a novel laccase gene from a white-rot fungus Polyporus grammocephalus TR16 and expression in Pichia pastoris.

AIMS: To isolate a novel laccase gene from white-rot fungus Polyporus grammocephalus TR16 and heterologous expression in Pichia pastoris. The characteristics of the heterologously expressed laccase are also studied. METHODS AND RESULTS: Anchored PCR and 3' RACE protocol were applied to obtain the full length of the laccase gene, which comprised 12 introns and an opening frame of 1769 bp. The deduced amino acid sequence of the laccase gene had an identity of 45-66% with the laccases reported previously. The cDNA was expressed in Pi. pastoris GS115 with native and α-factor secretion signal peptides. The laccase activity obtained with the native signal peptide is threefold higher than that obtained with the α-factor secretion signal peptide. The highest activity of the heterologously expressed laccase reached 893·3 U l(-1), with its molecular mass estimated to be 65·4 kDa by SDS-PAGE. The purified heterologously expressed laccase was stable at a pH range of 7·0-10·0. The optimum pH and temperature were 4·5 and 50°C, respectively; the K(m) value for ABTS (3-ethylbenzthiazoline-6-sulphonate) was 0·66 mmol l(-1). CONCLUSION: The novel laccase gene is cloned successfully and heterologously expressed in Pi. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel laccase gene isolated from a tropical fungus serves as a good source for pulp bleaching and wood processing.

Taxonomy of the major rhizomorphic species of the "Melanopus group" within Polyporaceae in Yasuní National Park, Ecuador.

Yasuní National Park in Ecuador is one of the most biodiverse places on earth. The fungi in this tropical rainforest are also diverse but have received little research attention. This research paper focuses on an important group of fungi in the family Polyporaceae and examines the genera Polyporus, Atroporus, and Neodictyopus that form aerial melanized cord-like structures called rhizomorphs. Phylogenetic analyses, macro and micromorphological descriptions of basidiomata and rhizomorphs, as well as cultural characterization were completed to better understand these ecologically important fungi. Here we describe four new species: Atroporus yasuniensis, Atroporus tagaeri, Neodictyopus sylvaticus, and Polyporus taromenane, and a new variety Polyporus leprieurii var. yasuniensis. The information presented in this study adds important new knowledge about the unusual rhizomorph producing fungi found in Yasuní National Park, Ecuador and other tropical rainforests.

Use of transcriptomic profiling to identify candidate genes involved in Polyporus umbellatus sclerotial formation affected by oxalic acid.

Polyporus umbellatus is a precious medicinal fungus. Oxalic acid was observed to affect sclerotial formation and sclerotia possessed more medicinal compounds than mycelia. In this study, the transcriptome of P. umbellatus was analysed after the fungus was exposed to various concentrations of oxalic acid. The differentially expressed genes (DEGs) encoding a series of oxidases were upregulated, and reductases were downregulated, in the low-oxalic-acid (Low OA) group compared to the control (No OA) group, while the opposite phenomenon was observed in the high-oxalic-acid (High OA) group. The detection of reactive oxygen species (ROS) in P. umbellatus mycelia was performed visually, and Ca2+ and H2O2 fluxes were measured using non-invasive micro-test technology (NMT). The sclerotial biomass in the Low OA group increased by 66%, however, no sclerotia formed in the High OA group. The ROS fluorescence intensity increased significantly in the Low OA group but decreased considerably in the High OA group. Ca2+ and H2O2 influx significantly increased in the Low OA group, while H2O2 exhibited efflux in the High OA group. A higher level of oxidative stress formed in the Low OA group. Different concentrations of oxalic acid were determined to affect P. umbellatus sclerotial formation in different ways.

Decrypting the Polyporus dictyopus complex: Recovery of Atroporus Ryvarden and segregation of Neodictyopus gen. nov. (Polyporales, Basidiomyocta).

Polyporus dictyopus, with a large number of heterotypic synonyms, has been traditionally considered a species complex, characterized by wide morphological variation and geographic distribution. Thus, neotropical specimens previously identified as P. dictyopus from Amazonia, Cerrado and Atlantic Forest biomes were studied based on detailed macro- and micromorphological examination and phylogenetic analyses, using distinct ribosomal and protein-coding genomic regions: the nuclear ribosomal internal transcribed spacer (nrITS), nuclear ribosomal large subunit (nrLSU), and RNA polymerase II second subunit (RPB2). Two unrelated generic lineages, each one represented by different species, are reported: Atroporus is recovered and re-circumscribed to include A. diabolicus and A. rufoatratus comb. nov.; Neodictyopus gen. nov. is proposed to accommodate N. dictyopus comb. nov. and two new species, N. atlanticae and N. gugliottae. Our study showed that at least five distinct species were hidden under the name P. dictyopus. Detailed descriptions, pictures, illustrations, and a key are provided for Atroporus and Neodictyopus species.

Phylogenetic constrains on Polyporus umbellatus-Armillaria associations.

It has been well established that some Armillaria species are symbionts of Polyporus umbellatus, However, little is known about the evolutionary history of P. umbellatus-Armillaria associations. In this research, we used an analysis based on the strength of the phylogenetic signal to investigate P. umbellatus-Armillaria associations in 57 sclerotial samples across 11 provinces of China. We isolated Armillaria strains from the invasion cavity inside the sclerotia of P. umbellatus and then phylogenetically analyzed these Armillaria isolates. We also tested the effect of P. umbellatus and Armillaria phylogenies on the P. umbellatus-Armillaria associations. We isolated forty-seven Armillaria strains from 26 P. umbellatus sclerotial samples. All Armillaria isolates were classified into the 5 phylogenetic lineages found in China except for one singleton. Among the 5 phylogenetic lineages, one lineage (lineage 8) was recognized by delimitation of an uncertain phylogenetic lineage in previous study. Results of simple Mantel test implied that phylogenetically related P. umbellatus populations tend to interact with phylogenetically related Armillaria species. Phylogenetic network analyses revealed that the interaction between P. umbellatus and Armillaria is significantly influenced by the phylogenetic relationships between the Armillaria species.

Transcriptomic analysis of the white rot fungus Polyporus brumalis provides insight into sesquiterpene biosynthesis.

Object of this study was to identify genes and enzymes that are involved in sesquiterpene biosynthesis in the wood rotting fungus, Polyporus brumalis. Sesquiterpenes, ß-eudesmane and ß-eudesmol, were produced by the mycelium of P. brumalis cultured in modified medium. However, theses final products were not observed when the fungus was grown in potato dextrose medium. We used next generation sequencing (NGS) to identify differentially expressed genes (DEGs) related to terpene metabolism. This approach generated 25,000 unigenes and 127 metabolic pathways that were assigned to Kyoto Encyclopedia Genes Groups (KEGG). Further analysis of samples from modified medium indicated significant upregulation of 8 unigenes involved in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways. These pathways generate isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP), which are precursors for the synthesis of sesquiterpenes. Furthermore, genes encoding germacrene A synthase, which facilitate the cyclization of FPP, were only differentially expressed in mycelium from fungi grown in modified medium. Our data provide a resource for studying the molecular mechanisms underpinning sesquiterpene biosynthesis and terpene metabolism.

Lentinoid and Polyporoid Fungi, Two Generic Conglomerates Containing Important Medicinal Mushrooms in Molecular Perspective.

Polyporoid and lentinoid fungi contain the important producers of substances having immunomodulatory, antitumoral, antiviral, and antihyperlipidemic effects. The discovery of several phylogenetic lines within the lentinoid-polyporoid continuum will help with target metabolomic analysis of species still not studied in pharmacological respects. The purpose of the present work was to increase a resolution in the lentinoid-polyporoid phylogenetic zone by means of selection of both the main representatives of Lentinus-related genera and poorly known/intermediate taxa such as Lentinus suavissimus, Neofavolus spp., and the resupinate part of Polyporus (genera Perenniporia and Pachykytospora) in the context of the basic structure of the Polyporales tree. The molecular phylogeny of highlighting all the polyporoid and lentinoid nodes was reconstructed using nLSU ITS rDNA and TEF datasets. The data obtained from ITS, TEF, and LSU coincide in support of core Polyporaceae of 10 clades corresponded to the generic level and 7 of these (Cerioporus, Cladomeris, Favolus, Lentinus, Neofavolus, Picipes, and Polyporus s.str.) contain generic units characterized by polyporoid or lentinoid morphotypes. The other 2 clades containing lentinoid taxa are outside the core Polyporaceae, namely Panus (Meruliaceae, Polyporales) and Neolentinus (Gloeophyllaceae, Gloeophyllales). A new genus, Picipes, is described and 25 new combinations are proposed.

Diversity of Polypore Bracket Mushrooms, Polyporales (Agaricomycetes), Recorded in Armenia and Their Medicinal Properties.

The list of polypore bracket mushrooms (Polyporales) recorded in Armenia is presented. The order Polyporales in Armenia is currently represented by 87 species (4 varieties) belonging to 47 genera. Information regarding the study of the medicinal properties (e.g., antifungal, antibacterial, mitogenic, regenerative, antioxidant, proteolytic) of genetically identified mycelial collections of several polypore species-mainly from the genera Daedalea, Fomes, Fomitopsis, Ganoderma, Laetiporus, Piptoporus, Polyporus, and Trametes-is reported, as well.

Development of Microsatellite Markers Derived from Expressed Sequence Tags of Polyporales for Genetic Diversity Analysis of Endangered Polyporus umbellatus.

A large scale of EST sequences of Polyporales was screened in this investigation in order to identify EST-SSR markers for various applications. The distribution of EST sequences and SSRs in five families of Polyporales was analyzed, respectively. Mononucleotide was the most abundant type, followed by trinucleotide. Among five families, Ganodermataceae occupied the most SSR markers, followed by Coriolaceae. Functional prediction of SSR marker-containing EST sequences in Ganoderma lucidum obtained three main groups, namely, cellular component, biological process, and molecular function. Thirty EST-SSR primers were designed to evaluate the genetic diversity of 13 natural Polyporus umbellatus accessions. Twenty one EST-SSRs were polymorphic with average PIC value of 0.33 and transferability rate of 71%. These 13 P. umbellatus accessions showed relatively high genetic diversity. The expected heterozygosity, Nei's gene diversity, and Shannon information index were 0.41, 0.39, and 0.57, respectively. Both UPGMA dendrogram and principal coordinate analysis (PCA) showed the same cluster result that divided the 13 accessions into three or four groups.