Analysis of expressed sequence tags (ESTs) from Lentinula edodes

The 1,031 expressed sequence tags (ESTs) from the basidiomycete Lentinula edodes were generated as a pilot experiment to see distribution of genes expressed in L. edodes. Among them, genes for hydrophobin, which are specifically found in filamentous fungi, were the most frequently obtained ESTs (33 times), suggesting that they are highly expressed in L. edodes. In addition to known hydrophobin 1 and 2 types, our analysis revealed the existence of novel types of hydrophobin, which we named hydrophobin 3, 4, and 5. The second and the third most highly obtained ESTs were phosphatidylserine decarboxylase and formate dehydrogenase, which were obtained eight and seven times, respectively. It should be noted that two important genes (argonaute and RNA-dependent RNA polymerase) involved in the RNAi pathway were found, suggesting a future application for gene knock-down by RNA interference. The 53 ESTs were identical with the sequences already reported in L. edodes. The 433 ESTs were found to show significant sequence similarity (E value <1 x 10(-5)) with the proteins reported (or predicted) in other species. In total, 387,952 bp were sequenced and registered in DDBJ/GenBank (accession number BJ998097-BJ999127).     

Morphological mutation of Lentinula edodes mycelium, particularly detectable in the dikaryotic state

A morphological mutation particularly detectable in the dikaryotic state was found in Lentinula edodes. The mutant dikaryon was readily distinguishable from the normal dikaryon by the irregularly branched short hyphae, very slow hyphal growth, and sparse aerial hyphae. Genetic analysis revealed that expression of this mutation was controlled by a single recessive gene, mor-13. Linkage analysis showed that the mor-13 was not linked to either the incompatibility factors (A and B) or the five kinds of mor genes that were segregated independently of each other in a previous study. Contribution no. 380 from the Tottori Mycological Institute     

A new biological function of Shiitake mushroom,Lentinula edodes, in a myco-heterotrophic orchid,Erythrorchis ochobiensis

The biological function ofLentinula edodes in a myco-heterotrophic orchid,Erythrorchis ochobiensis was examined, using one local variant each from Japan (JPN), Papua New Guinea (PNG) and New Zealand (NZ). All variants induced seed germination: PNG and NZ isolates were effective at 25°C and JPN isolate showed the highest germination rate at 30°C. Germinated seeds developed into plants and formed normal endomycorrhizas. Hence, it is concluded thatL. edodes has a perfect symbiotic potential withE. ochobiensis, though it has not been observed in the root of the orchid in the field.     

Genetic analysis of nuclear ribosomal DNA of Lentinula edodes

Genetic analysis of nuclear ribosomal DNA (rDNA) of Lentinula edodes was carried out using rDNA restriction fragment length polymorphisms (RFLPs) as genetic markers. Two compatible monokaryotic strains that differed in the endonuclease digestion patterns of their rDNA were used. The dikaryotic strain established by crossing them produced mixed RFLP patterns. Single-spore isolates derived from the dikaryotic strain showed three types of rDNA RFLP patterns: either one of the two parental types or a mixed type. From the frequency of the mixed type, the recombination value of rDNA tandem repeats was calculated to be 31.4%. Linkage analysis between rDNA and two incompatibility factors (A and B) revealed that rDNA was not linked to either factor. The rDNA genotypes did not affect mycelial growth among the single-spore isolates.     

A method for extraction of total RNA fromPinus radiata and other conifers

Carbohydrates, polyphenolic compounds, terpenoids and tannins interfere with the extraction of intact, uncontaminated total RNA from conifers. A method for extraction of total RNA fromPinus radiata is described. This method uses cesium trifluoroacetate in the ultracentrifugal separation of RNA to overcome the problems of co-purification of contaminating secondary metabolites.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Detection and distribution of six linear mitochondrial plasmids in the shiitake mushroom,Lentinula edodes

The presence of plasmids was surveyed in 90 wild isolates ofLentinula edodes collected from geographically different world regions. DNA plasmids of different sizes were found in about 80% of the isolates. The plasmids detected were of six kinds, designated as pLE1 (9.0 kb), pLE2 (11.1 kb,=pLLE1 described by other authors), pLE3A (9.8 kb), pLE3B (10.8 kb), pLE3C (12.1 kb), and pLE3D (12.3 kb). Hybridization analysis suggested that pLE1 and pLE2 were distinct plasmid types of different homology groups to each other, and the four other plasmids were variant types belonging to a third homology group. These plasmids had no homology with their host's and non-host's nuclear and mitochondrial genome DNAs. Restriction analysis and electron microscopy indicated that the plasmids are linear in form. Since all six plasmids were transmitted uniparentally in sexual crosses and were consistently associated with the DNA preparations from mitochondria fractionated from mycelia of representative isolates, they were suggested to be located in mitochondria, similar to many other known fungal DNA plasmids. Geographically, pLE1 and pLE2 were widely distributed in natural populations ofL. edodes, while the remaining four plasmids were uniquely present in delimited natural populations. Contribution No. 322 from the Tottori Mycological Institute.     

Viability and mushroom production of Lentinula edodes and L. boryana strains (Fungi: Basidiomycetes) after cryogenic storage of spawn stocks

The viability of two strains of Lentinula edodes and two of L. boryana under cryogenic storage during 1 week has been studied from the evaluation of five contact periods (1, 1.5, 2, 3 and 5 h) of the cryoprotector, glycerol 10% (v/v), with the mycelium. On average, 99.25% of samples were recovered, 1.5 h being the best contact period. Afterwards, samples of the strains, before and after the cryogenic process, were cultivated at a pilot plant using a mix of Carpinus carolineana sawdust, rice bran and sorghum grains as substrate. The evaluation parameters were: days of incubation, primordia initiation, number of flushes, fruiting body sizes and biological efficiency (EB). Only L. edodes developed carpophores. On average, 3–4 flushes were obtained, which reached EB of 67.1 ± 30.7 to 74.7 ± 24.5 with no statistical differences detected between the yields. The majority of fructification sizes ranged from 5 to 14.9 cm. Morphological differences between the samples before and after the treatment were not observed.     

A reliable method for extraction of RNA from various conifer tissues

Summary A simple and efficient procedure suitable for extraction of high-quality RNA from cultured conifer tissues, somatic embryos, zygotic embryos, needles, stem and root tissues was developed. It produced from 100 g up to 700 g total RNA per gram tissue dependent on the types of tissues used. RNA quality was estimated by spectrophotometry, agarose gel electrophoresis, in vitro translation of mRNA, cDNA synthesis and Northern blot analysis. The method also worked well with Arabidopsis thaliana and tobacco tissues.Abbreviations CTAB cetyltrimethylammonium bromide - DEPC diethylpyrocarbonate - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Cloning and Characterization of the Xyn11A Gene from <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Hemicellulose represents a rich source of biomass that can be converted into useful chemical feedstocks. One of the main components of hemicellulose is xylan, a polymer of xylose residues. Xylanase enzymes that hydrolyze xylan are therefore of great commercial interest. We have cloned a gene (xyn11A) that encodes a 283-amino acid xylanase enzyme from the fungus Lentinula edodes. The enzyme has a pI of 4.6 and belongs to the highly conserved glycosyl hydrolase family 11. The xylanase gene was cloned into a Pichia pastoris expression vector that secretes active enzyme into both solid and liquid media. The optimal reaction conditions were at pH 4.5 and 50°C. The enzyme had a K_m of 1.5 mg/ml and a V_max of 2.1 mmol/min/mg. Xyn11A produced primarily xylobiose, xylotriose, and xylotetraose from a birchwood xylan substrate. This is the first report on the cloning of a hemicellulase gene from L. edodes.     

A simple and effective method for isolating RNA from alfalfa pollen

Extraction of RNA from alfalfa pollen using conventional grinding devices resulted in low yields of degraded RNA; degradation appeared to be related to the length of time required to break open the pollen grains with such devices. A glass syringe was modified to provide the restricted, shearing environment necessary for rapidly rupturing this type of tissue. This apparatus would be useful for extracting a variety of molecules from pollen of any species, but is particularly valuable for species which produce low amounts of relatively small pollen grains.     

A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 °C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.Communicated by G. Antranikian     

Induction of apoptosis in cultured cells by extracts from shiitake (Lentinula edodes) mycelial culture broth

Extracts fromshiitake (Lentinula edodes) mycelial culture broth, by an organic solvent ethyl acetate, inhibited the proliferation of cultured cells. At lower concentrations (1.25–15 μg/ml), this inhibition, measured by the MTT assay, was dose- and cell line-dependent. Inhibition of tumor cells, such as Caski, SiHa, HeLa, HP-1 and A375, byL. edodes-436 extracts was stronger than inhibition of normal cells (3T3). At 20 μg/ml, the extracts induced changes in cell shape, DNA-fragmentation and the activation of caspase-3. The extracts also inhibited the binding of E2F protein to its promoter. The results suggest that extracts ofL. edodes culture broth contain substances that have the ability to induce apoptosis in the cultured cells.     

An effective, rapid and simple method for total RNA extraction from bacteria and yeast

In this work, we describe a rapid and simple method for total RNA extraction from bacteria and yeast. The method allows for the acquirement of high RNA yields while avoiding the use of phenol or other toxic reagents and is less expensive than other methods previously described. The extracted RNA is suitable for applications such as RT-PCR, Northern blot hybridization and low molecular weight RNA (LMW RNA) electrophoresis.     

Effect of liquid mycelial culture used as a spawn on sawdust cultivation of shiitake (Lentinula edodes)

Mycelium of shiitake (Lentinula edodes) was cultured continuously in liquid medium. The liquid culture was carried out for the production of liquid spawn in the cultivation of this mushroom on synthetic sawdust substrate, and its performance was compared with that of the solid spawn. The initial colonization in culture bags was faster with the solid spawn than with the liquid spawn, but after this stage CO₂ production was higher with the liquid spawn than with the solid spawn. For harvesting sufficient amount of good quality mushrooms, 120 d of incubation in bags was needed with the solid spawn, but this was reduced to 90 d for the sawdust blocks using liquid spawn of less than 50 d old. If continuous culture of the liquid spawn was prolonged over 50 d, immature fruit-bodies or their initials formed during the period of bag incubation. The solid subcultures of the liquid spawns retained the fruiting characteristics acquired in the liquid culture. Liquid culture could be a useful tool for breeding of mushrooms.     

一种适用范围广的总RNA提取方法

介绍一种RNA提取方法,该方法以SDS、氯仿和Tris苯酚为主要提取试剂,以LiCl和乙醇为RNA沉淀试剂。分别以柽柳(木本植物)、星星草(草本植物)、天牛（昆虫）、酿酒酵母和白腐菌（真菌）为RNA提取材料,用该方法成功地提取出了它们的总RNA。获得的RNA条带清晰,A₂₆₀/A₂₈₀ 在1.8以上。通过对LiCl和乙醇沉淀RNA的效果分析表明,该方法可在10 min内完全沉淀RNA,同时也可以同时获得纯度较高的DNA。提取的RNA质量可满足cDNA文库构建,基因芯片探针标定和RT-PCR等对RNA质量要求较高的分子生物学操作,说明这是一种应用范围广的RNA提取方法。     

NMR characterization of the polysaccharidic fraction from Lentinula edodes grown on olive mill waste waters

A high-field NMR study of the polysaccharidic fraction extracted from Lentinula edodes mycelium grown on olive mill waste waters is reported. Diffusion-ordered NMR spectroscopy (DOSY) was applied to the polysaccharidic fraction. The results showed the presence of two polysaccharides of different sizes, whose structures were revealed using one- and two-dimensional NMR techniques. These two polysaccharides were identified as xylan and lentinan.