Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes.

Use of P450 cytochrome inhibitors in studies of enokipodin biosynthesis

Enokipodins A, B, C, and D are antimicrobial sesquiterpenes isolated from the mycelial culture medium of Flammulina velutipes, an edible mushroom. The presence of a quaternary carbon stereocenter on the cyclopentane ring makes enokipodins A-D attractive synthetic targets. In this study, nine different cytochrome P450 inhibitors were used to trap the biosynthetic intermediates of highly oxygenated cuparene-type sesquiterpenes of F. velutipes. Of these, 1-aminobenzotriazole produced three less-highly oxygenated biosynthetic intermediates of enokipodins A-D; these were identified as (S)-(-)-cuparene-1,4-quinone and epimers at C-3 of 6-hydroxy-6-methyl-3-(1,2,2-trimethylcyclopentyl)-2-cyclohexen-1-one. One of the epimers was found to be a new compound.

Removal of Cu2+ ions from aqueous solution by the abandoned mushroom compost of Flammulina velutipes.

The abandoned mushroom compost of Flammulina velutipes, a cheap and easy by-product to get, was used as biosorbent for removing copper ions from aqueous solution. Batch experiments were carried out to investigate the effect of contact time, solution pH, biomass dosage, initial concentration of Cu2+ ions and temperature on biosorption efficiency. The maximum sorption capacity could be reached at pH 5.0 in 60 min. Langmuir, Freundlich, and Redlich- Peterson isotherm models were used to fit the experimental data and their model parameters were evaluated. The calculated qm based on Langmuir equation was 35.608 mg g-1 at 288 K, 48.711 mg g-1 at 298 K, and 42.330 mg g-1 at 308 K, respectively. The kinetics were discussed by pseudo- first order and pseudo- second order models, and the result showed that the latter was more suitable. The thermodynamics of biosorption was also investigated, and the biosorption process was feasible, spontaneous and endothermic in nature.

Cloning and Characterization of a Novel Laccase Gene, fvlac7, Based on the Genomic Sequence of Flammulina velutipes

Laccases (EC 1.10.3.2) are copper-containing polyphenol oxidases found in white-rot fungi. Here, we report the cloning and analysis of the nucleotide sequence of a new laccase gene, fvlac7, based on the genomic sequence of Flammulina velutipes. A primer set was designed from the putative mRNA that was aligned to the genomic DNA of F. velutipes. A cDNA fragment approximately 1.6-kb long was then amplified by reverse transcriptase-PCR using total RNA, which was subsequently cloned and sequenced. The cDNA sequence of fvlac7 was then compared to that of the genomic DNA, and 16 introns were found in the genomic DNA sequence. The fvlac7 protein, which consists of 538 amino acids, showed only 42~51% identity with 12 different mushroom species containing two laccases of F. velutipes, suggesting the fvlac7 is a novel laccase gene. The first 25 amino acids of Fvlac7 correspond to a predicted signal sequence, four copper-binding sites, and four N-glycosylation sites. Fvlac7 cDNA was heterologously overexpressed in an Escherichia coli system with an approximate expected molecular weight of 60 kDa.

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/microg of DNA in 1 x 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

Agrobacterium-mediated Transformation of the Winter Mushroom, Flammulina velutipes

Flammulina velutipes was transformed efficiently by Agrobacterium-mediated transformation system. The transformation frequency was about 16% with the gill tissues of the fungal fruiting body. Southern hybridization and genetic analysis suggest that the introduced DNA was inserted onto different locations of the fungal genome, and inherited stably to the next generation via basidiospores. Transformation or gene tagging with Agrobacterium T-DNA based vector should be useful for wide ranges of genetic or molecular biological studies of the mushroom.

Molecular Marker Related to Fruitbody Color of Flammulina velutipes

White and brown strains of Flammulina velutipes were inter-crossed. All F1 showed light-brown fruitbody, suggesting that a gene for the brown fruitbody was incompletely dominant against the white one. And backcross experiment showed that more than two genes were involved in color determination. To isolate a molecular marker linked to fruitbody color, a set of primers was designed from a sequence of clones derived by a bulked segregant analysis. These markers showed a specific band which co-segregated with brown fruitbody forming strains.

Breeding of Flammulina velutipes Strains Adaptable to Elevated-temperature

Winter mushroom, Flammulina velutipes, needs low temperature during its cultivation. To save on farm costs, especially during summer, a strain adaptable to a higher or elevated-temperature must be developed. At the start of breeding program, parental strains which could endure high temperature were obtained. Seuenty four dikaryotic strains were collected and divided into four groups according to the nature of temperature. They also had different fruiting temperature. Finally we selected three brown strains ASI 4048, 4057 and 4072, and collected their spores. These selected strains can germinate even at a high temperature of 32degrees C, which were dramatically higher than the other strains. Based on these results, the new white strain adapted to mid-temperature by backcross mating was developed. Molecular markers were applied to select white fruit-body producing strains without cultivation. They showed a specific band which co-segregated with brown fruitbody forming strains in BC1F1 progenies. Selected white strains were tested under several elevated temperature conditions.

DETECTION OF FLAMMULIN WITH IMMUNOBLOT / 微生物学通报

Flammulin, an anti-tumor protein, was purified from the aqueous extract of basidiomes of Flammulina Velutipes. Purified flammulin emulsified with Freund's adjuvant was injected subcutaneously into New Zealand white rabbits. After several immune enhancements, these animals were bled and sera were separated. Antiserum against flammulin in Western blots were applied to determine if flammulin be present in the liquid state culture or fruiting body. The result showed that anti-flammulin serum could recognize the aqueous extract of fruiting body in SDS-PAGE gels under the reducing conditions, no flammulin was detected in mycelia of Flammulina Velutipes.

ISOLATION AND PHARMACOLOGICAL ACTIVITIES OF AN ANTITHROMBUS PROTEIN FROM FLAMMULINA VELUTIPES / 中国药理学通报

A protein designated as Flammulina Velutipes (FV) protein was isolated from the cultured mycelium of FV and purified to homogeneity by CM cellulose CM 52 and gel filtration chromatography. The molecular weight of FV protein were found to be 1.75?10~4u with pI 4.3. Amino acid analysis of FV protein indicated the presence of 14 kinds of amino acids, especially rich in Glu, Asp and Thr.Potent inhibitory effect of FV protein on thrombus formation, in the blood was observed in vitro when rats were given intravenously. The length of thrombus was shortened and the dry weight of the thrombus decreased significantly at the dose of 7.5?10~(-7) mol/kg. Thrombus formation was completely inhibited at the dose of 10~()-8 mol/kg. The IC_(50) was found to be 4.4?10~(-7) mol/kg. FV protein was shown to inhibit collegen induced aggregation of rat platelets both in vitro and in vivo. Its concentration for complete inhibition in vitro was 7.5?10~(-5) mol/L. The IC_(50) was found to be 4?10~(-5)mol/ L. However, FV protein dose of 1.25?10~(-5) mol/kg was required for 60% inhibition in vivo

EXPERIMENTAL RESEARCH ON THE ANTIFATIGUE EFFECT OF FLAMMULINA VELUTIPES / 营养学报

The present paper reports a systematic research of the antifatigue effect of Flammulina velutipes. The antifatigue effect was judged by the examination of serum lactate dchydrogenase activity, level of blood lactic acid, serum urea nitrogen, muscle and liver glycogen,The experiments indicated that feeding Flammulina velutipes to mice for several days the lactate dehydrogenase activity, muscle and liver glycogeu levels were significantly higher than that of the control. After exercise, the levels of blood lactate and serum urea nitrogen were significantly lower than those of control. After exercise, the recovery rate of lactic acid was much faster than that of control.From the above results, we concluded the Flammulina velutipes may have significant effect on the capability of adaptation to heavy exercise and prevention or elimination of fatigue after exercise.