杨树菇凝集素AAVP具有抗病毒和促进菌丝分化功能

杨树菇凝集素 (AAVP)是一种新的真菌凝集素 .用半叶法检测证明 ,AAVP具有抑制烟草花叶病毒 (TMV)侵染的活性 .等电聚焦法证实了AAVP可以与TMV的外壳蛋白结合 .用滤纸圆片法检测 ,AAVP对 3种植物病原真菌没有抑制作用 .AAVP滴加在菌丝的表面 ,显著地促进了杨树菇和毛木耳的菌丝分化 ,促进子实体的形成 .Western印迹表明 ,AAVP存在于菌丝、菌柄和菌盖等组织中 ;假猴头 ,香菇 ,草菇 ,杏孢菇 ,灵芝等大型真菌的子实体中存在着多种与AAVP的抗血清有交叉反应的蛋白质 .大多数大型真菌中可能存在着与AAVP具有相似血清学特征的蛋白质家族 ,在防御反应及菌丝分化等多种生理过程中发挥重要的作用     

A lectin with mycelia differentiation and antiphytovirus activities from the edible mushroom Agrocybe aegerita

A lectin named AAL has been purified from the fruiting bodies of the edible mushroom Agrocybe aegerita. AAL consisted of two identical subunits of 15.8 kDa, its pI was about 3.8 determined by isoelectric focusing, and no carbohydrate was discerned. Being treated by pyrogultamate aminopeptidase, the blocked N-terminus of AAL was sequenced as QGVNIYNI. AAL agglutinated human and animal erythrocytes regardless of blood type or animal species. Its hemagglutinating activity was unaffected by acid or alkali treatment and demetalization or addition of divalent metals Mg(2+), Ca(2+) and Zn(2+). AAL was toxic to mice: its LD50 was 15.85 mg per kilogram body weight by intraperitoneal injection. In this study, two novel activities of AAL were proved. It showed inhibition activity to infection of tobacco mosaic virus on Nicotiana glutinosa. The result of IEF suggested that AAL attached to TMV particles. Mycelia differentiation promotion was the other interesting activity. AAL promoted the differentiation of fruit body primordia from the mycelia of Agrocybe aegerita and Auricularia polytricha. AAL antiserum was prepared and immunologically cross-reactived with several proteins from five other kinds of mushrooms. These results suggested that AAL probably was a representative of a large protein family, which plays important physiological roles in mushroom.     

糙皮侧耳生长发育过程中漆酶基因家族的表达研究

漆酶参与木质素的降解和食用菌的生长发育。为了明晰漆酶基因在糙皮侧耳生长发育过程中的作用,本文采用real-time PCR检测了11条漆酶基因及Lacc2的小亚基sspoxa3在糙皮侧耳生长发育不同阶段的表达。其中lacc6和sspoxa3在整个生长发育阶段表达量均较高;lacc12随着原基的分化和子实体的形成大量表达,与成菇过程有关。lacc4,lacc7和lacc11在原基分化期高表达,与原基的分化有关。lacc2,lacc3和lacc8在成熟子实体阶段表达量显著上升,与子实体的分化和成熟有关。     

信息素信号通路基因在斑玉蕈生长发育过程中的差异表达

为了更清楚地了解斑玉蕈菌丝成熟、原基形成和子实体发育的过程,本研究对不同菌丝培养时期的栽培瓶进行出菇实验,并对其不同培养时期和生长发育关键时期的信息素通路基因进行差异表达分析,以期揭示信息素信号通路基因参与调节斑玉蕈菌丝的生长、子实体形成和发育的作用。研究结果表明：斑玉蕈菌丝培养40-80d过程中,子实体产量呈上升的趋势,说明菌丝的成熟程度对产量会产生重要影响。对斑玉蕈基因组中的信息素信号通路基因进行分析鉴定共获得了8个关键基因。信息素通路基因差异表达分析表明：在菌丝培养40-80d过程中,大部分信息素信号通路基因在第60天时表达量最高,其中ste20、cdc24和ste12上调了4-20倍,而在第80天出现下降。从菌丝恢复到扭结形成原基和子实体发育的过程中,大多数基因在原基时期表达量最高,其中ste20、cdc24、ste11和ste12表达量上调最为显著,在子实体成熟期这些基因表达量下降。因此,这说明在菌丝营养生长过程中,在第60天菌丝细胞增殖生长最为旺盛,而在第80天菌丝细胞基本停止生长,菌丝也逐渐达到成熟。同时,在菌丝生殖生长过程中,斑玉蕈持续地上调信息素通路基因表达使菌丝细胞不断地分裂增殖,从而使新生的菌丝扭结形成原基,其中ste3、ste20、cdc24、ste11和ste12基因可能对斑玉蕈菌丝细胞的分裂增殖和诱导子实体形成起到关键的作用。     

金针菇信息素信号通路基因在子实体发育过程中的差异表达

【背景】子实体是食用菌的主要商品部位,也是真菌生殖生长的重要结构,其发育受到多种信号途径的调控。【目的】以金针菇(Flammulina filiformis)为材料,对转录组和基因组数据的信息素信号通路基因进行分析获得差异表达的基因,并对其在菌丝生长和子实体发育过程中的表达情况进行分析,以期为研究食用菌子实体发育提供参考。【方法】基于已有的金针菇基因组数据,注释了金针菇信息素信号通路。进一步通过转录组测序鉴定了该通路中参与金针菇子实体发育的关键基因,并对关键基因进行荧光定量PCR验证。【结果】cdc24和ste12基因在子实体发育不同时期的5个样品(原基、伸长期菌柄、伸长期菌盖、成熟期菌柄和成熟期菌盖)中的表达具有显著差异,使用荧光定量PCR技术进行验证与上述结果一致。【结论】cdc24和ste12这2个关键基因可能参与了金针菇子实体发育过程中的组织分化调控机制。     

Crystallization and preliminary crystallographic studies of an antitumour lectin from the edible mushroom Agrocybe aegerita

An antitumour lectin named AAL has been purified from the fruiting body of edible mushroom Agrocybe aegerita. In addition to having a distinct bioactivity, AAL shows strong inhibition effects on human and mouse tumour cells. It has been shown that AAL exerts its antitumour effects via apoptosis-induction. AAL and AAL-lactose complex have been crystallized and their diffraction data were collected with resolution of 2.6 A and 3.0 A, respectively. Both crystals belong to space group P 6122 with unit cell parameters a = 123.98 A, b = 123.98 A, c = 56.86 A, alpha= beta=90 degrees, gamma = 120 degrees and a = 123.69 A, b = 123.69 A, c = 56.64 A, alpha = beta = 90 degrees, gamma = 120 degrees, respectively.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Primary structure of a fucose-specific lectin obtained from a mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) is a protein composed of two identical subunits having no carbohydrate chain and shows sugar-binding specificity for L-fucose. Full-length cDNA encoding for the lectin has been isolated from a lambda gt11 library, screened with an antiserum directed against AAL. The cDNA clone contained 1,370 nucleotides and an open reading frame of 939 nucleotides encoding 313 amino acids. The amino-terminal sequence (residues 1-30) of the lectin isolated from the mushroom coincided with the deduced amino acid sequence starting from proline at the 2nd residue, indicating that the mature AAL consists of 312 amino acids. Its molecular weight is calculated to be 33,398. The deduced amino acid sequence shows that AAL includes six internal homologous regions, and has considerable homology with a hemagglutinin from a Gram-negative bacterium, Myxococcus xanthus, which forms a fruiting body. No significant homology was observed with higher plant or animal lectins. The recombinant AAL produced by Escherichia coli JM109 carrying the AAL expression plasmid pKA-1 [Fukumori, F. et al. (1989) FEBS Lett. 250, 153-156] was purified from the cell lysate by affinity chromatography using a fucose-starch column, and hundreds of milligrams of the lectin was obtained. The recombinant lectin showed the same biochemical characteristics and sugar binding specificity as did the natural AAL.     

Soil fungal communities in a Castanea sativa (chestnut) forest producing large quantities of Boletus edulis sensu lato (porcini): where is the mycelium of porcini?

A study was conducted in a Castanea sativa forest that produces large quantities of the edible mushroom porcini (Boletus edulis sensu lato). The primary aim was to study porcini mycelia in the soil, and to determine if there were any possible ecological and functional interactions with other dominant soil fungi. Three different approaches were used: collection and morphological identification of fruiting bodies, morphological and molecular identification of ectomycorrhizae by rDNA-ITS sequence analyses and molecular identification of the soil mycelia by ITS clone libraries. Soil samples were taken directly under basidiomes of Boletus edulis, Boletus aestivalis, Boletus aereus and Boletus pinophilus. Thirty-nine ectomycorrhizal fungi were identified on root tips whereas 40 fungal species were found in the soil using the cloning technique. The overlap between above- and below-ground fungal communities was very low. Boletus mycelia, compared with other soil fungi, were rare and with scattered distribution, whereas their fruiting bodies dominated the above-ground fungal community. Only B. aestivalis ectomycorrhizae were relatively abundant and detected as mycelia in the soil. No specific fungus-fungus association was found. Factors triggering formation of mycorrhizae and fructification of porcini appear to be too complex to be simply explained on the basis of the amount of fungal mycelia in the soil.     

CWI和HOG信号通路基因在斑玉蕈不同发育阶段的表达模式

为了更清楚地了解MAPK信号通路中的细胞壁完整性信号通路（cell wall integrity,CWI）和高渗透压甘油（high-osmolarity glycerol pathway,HOG）信号通路对斑玉蕈菌丝成熟、原基形成和子实体发育过程的影响及调节作用,对MAPK信号通路中的CWI和HOG信号通路基因在斑玉蕈不同菌丝培养时间（40、60、80和100d）和不同生长发育关键时期（24h、菌丝恢复期、菌丝转色期、原基期和子实体期）的表达模式进行分析,以期揭示这两条信号通路基因参与调节斑玉蕈菌丝的生长、子实体的形成和发育的作用。在斑玉蕈的CWI和HOG信号通路中经分析鉴定一共获得了15个关键基因。CWI信号通路基因表达分析表明：在菌丝培养的40-100d的过程中,大部分CWI信号通路基因在第60天时表达量最高,其中rho1、ssk1、ssk2和ste20的基因表达量上调了2-5倍,在第80-100天时出现持续下降。在HOG信号通路中的大部分基因也在菌丝培养的第60天表达量达到最高。其中sho1、ste20、ssk1和ssk2基因的表达量上调最为显著,而hog1基因的表达量在菌丝培养的第40-100天呈持续下降。子实体形成过程中两条通路的大部分基因在原基形成时期表达量最高,而在子实体时期表达量下调。其中HOG信号通路中的ssk2基因表达量上调最为显著。以上结果说明在菌丝生长过程中第60天时菌丝细胞生长增殖最为旺盛,而在第80天开始菌丝细胞基本开始停止生长,菌丝也逐渐达到成熟。同时在菌丝增殖生长过程中,斑玉蕈持续地上调CWI信号通路基因的表达来调控菌丝细胞壁的完整性,从而控制菌丝细胞壁的形成。其中bck1、mkk1和slt2基因可能对斑玉蕈菌丝细胞的分裂增殖和细胞壁的形成以及诱导子实体形成起到关键作用。     

Molecular character of the recombinant antitumor lectin from the edible mushroom Agrocybe aegerita

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.     

Characterization, occurrence, and molecular cloning of a lectin from Grifola frondosa: jacalin-related lectin of fungal origin

A lectin named GFL was isolated from the fruiting body of the basidiomycete mushroom Grifola frondosa, which belongs to Aphyllophorales. The lectin had a molecular mass of 24 kDa on SDS-PAGE. The hemagglutinating activity of GFL was not inhibited by any monosaccharide, and inhibited only by porcine stomach mucin so far as tested. The occurrence of GFL was studied at three stages during fruiting body formation. The largest quantity of hemagglutinating activity was found in the fruiting body, and lesser amounts in the mycelial mat and the primordium. The 24-kDa band of GFL was found at all three stages, and the band-intensity corresponded to the level of activity in each sample. By cloning and sequencing the GFL-cDNA, the primary structure of this lectin was determined. GFL is composed of 181 amino acids, having no signal peptide. The amino acid sequence was found to be homologous to those of so-called jacalin-related plant lectins, suggesting that GFL is the first example of a jacalin-related lectin of fungal origin.     

蛹虫草对锌的耐性与富集特征

在培养基内添加不同量的锌,研究其对蛹虫草子实体的形成、子实体和菌丝体生物量、子实体多糖含量和葡萄糖含量的影响,以及蛹虫草子实体和菌丝体对锌的富集能力。结果表明锌对上述各项都有影响。液体培养条件下,锌浓度在453906mg/L范围内可以促进菌丝体生长,锌浓度超过4077mg/L时,菌丝生长受到抑制。培养基锌的浓度在4077mg/L以下时,蛹虫草菌丝体锌的富集量随着液体培养基锌浓度的提高而提高。固体培养条件下,锌含量在226453mg/kg范围内可以促进蛹虫草子实体生长,并且在此含量范围内,蛹虫草子实体中葡萄糖含量较高。培养基锌含量在680906mg/kg时,子实体多糖含量较高。培养基锌含量在2038mg/kg以下时,蛹虫草子实体中锌的富集量随着培养基锌含量的提高而提高,在培养基锌含量为2038mg/kg时,子实体中锌的含量达到28570mg/kg(干重)。     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

Molecular cloning of developmentally specific genes by representational difference analysis during the fruiting body formation in the basidiomycete Lentinula edodes

To understand molecular mechanisms of the fruiting body development in basidiomycetes, we attempted to isolate developmentally regulated genes expressed specifically during the fruiting body formation of Lentinula edodes (Shiitake-mushroom). cDNA representational difference analysis (cDNA-RDA) between vegetatively growing mycelium and two developmental substages, primordium and mature fruiting body, resulted in an isolation of 105 individual genes (51 in primordium and 54 in mature fruiting body, respectively). A search of homology with the protein databases and two basidiomycetous genomes in Phanerochaete chrysosporium and Coprinopsis cinerea revealed that the obtained genes encoded various proteins similar to those involved in general metabolism, cell structure, signal transduction, and responses to stress; in addition, there were apparently several metabolic pathways and signal transduction cascades that could be involved in the fruiting body development. The expression products of several genes revealed no significant homologies to those in the databases, implying that those genes are unique in L. edodes and the encoding products may possess possible functions in the course of fruiting body development. RT-PCR analyses revealed that 20 candidates of the obtained genes were specifically or abundantly transcribed in the course of the fruiting body formation, suggesting that the obtained genes in this work play roles in fruiting body development in L. edodes.