低温胁迫诱导肺形侧耳合成麦角甾醇的通路分析

本文以肺形侧耳栽培菌株X57为研究对象,对其低温胁迫时期进行转录组分析,发现差异基因显著地富集到麦角甾醇合成通路。根据麦角甾醇合成通路的相关信息,在肺形侧耳中筛选出该通路的17个基因,并推测了肺形侧耳的麦角甾醇合成通路。利用荧光定量研究了X57栽培袋在低温处理下该通路上各个基因的表达,结果显示随着低温胁迫时间的增加,该通路上大部分基因的表达量持续上升,且差异显著。检测栽培袋中菌丝麦角甾醇含量发现,低温胁迫显著提高麦角甾醇的含量,与RNA‐Seq分析结果一致。此外,以无需打冷也能整齐出菇的野生菌株X1为对照,比较X57与X1的麦角甾醇基因对冷胁迫的响应情况,探究麦角甾醇合成通路是否在肺形侧耳低温诱导形成原基时期具有关键性作用。将X57与X1菌株置于PDA平板上培养并进行低温胁迫,利用荧光定量PCR对各个基因表达进行验证,结果显示两个菌株的麦角甾醇合成通路相关基因均对低温胁迫有响应,X57低温胁迫时通路中绝大多数基因的表达量上升,且大部分基因表达量提高2倍以上;X1菌株的通路中的表达量变化较小。由此推测麦角甾醇通路在肺形侧耳变温结实中低温刺激形成原基有一定作用,为深入研究肺形侧耳低温胁迫调控分子机理提供了重要的基础。     

肺形侧耳线粒体基因组多态性鉴定

线粒体是能量转化与ATP形成的重要场所,已有多种食用真菌的线粒体基因组被相继组装注释,但肺形侧耳的线粒体基因组鲜有报道。本研究对野生肺形侧耳X2菌株的线粒体基因组进行组装并注释,由野生菌株X2与主栽菌株JX线粒体大片段差异序列,构建分子标记来鉴别野生和主栽肺形侧耳菌株。结果显示X2的线粒体基因组为大小75 709bp的闭合环状结构,含有rRNA的大小亚基基因,25个负责携带氨基酸的tRNA基因以及14个常见蛋白编码基因,在cox1基因上含有9个内含子。内含子主要为IB型,包含LAGLIDADG_1 superfamily、GIY-YIG_Cterm superfamily保守结构域。位于菌株X2与JX内含子和基因间区上的大片段差异序列,是造成种内线粒体多态性的主要因素。根据两菌株的差异片段构建系统进化树的结果显示,内含子、rnl-trnX、trnD-atp6序列能够将两株野生菌株和其他主栽菌株区分开,可用于野生肺形侧耳种内鉴定,而且trnD-atp6效果最佳。     

我国肺形侧耳栽培菌株交配型分析

为分析我国栽培肺形侧耳的遗传多样性,对肺形侧耳菌株的交配型进行测定,测试的肺形侧耳菌株包括俗称为秀珍菇和凤尾菇的菌株.结果 显示,我国栽培的秀珍菇与凤尾菇菌株间可以发生性亲和,属于同一生物学物种,但它们之间的交配型特异性低,36个菌株中只含4种A交配型和3种B交配型,暗示我国栽培的肺形侧耳种质资源基因匮乏.     

肺形侧耳低温胁迫时期的转录组分析

本文以肺形侧耳栽培菌株X57为研究对象,利用高通量RNA测序技术对4℃低温处理0h、6h和12h后的肺形侧耳进行基因表达分析来探讨肺形侧耳低温应答机制。分析结果筛选出低温处理6h差异表达基因742个,其中上调表达基因374个,下调表达基因368个;低温处理12h差异表达基因1 489个,其中上调表达基因占53%,下调表达基因占47%。Gene Ontology（GO）功能聚类分析表明,差异表达基因主要富集在结合、催化分子功能组和代谢过程、细胞过程生物学过程中。KEGG功能富集分析结果显示,差异基因主要富集在氨基酸、核糖体和类固醇生物合成,及氮类物质代谢的通路上,富集到与低温胁迫相关通路MAPK signaling pathway-yeast上的基因表达随低温处理时间的增加呈上调趋势。已报道HOG-MAPK通路是MAPK途径研究较为明确、信号传递单一的真菌低温胁迫中重要的通路,本文运用生物信息学软件构建肺形侧耳的HOG-MAPK通路,并利用荧光定量PCR对相关基因表达进行验证,结果显示以低温处理0h为参照,随着低温胁迫时间增加通路上大部分基因的表达量持续上升,且差异显著,与RNA‐Seq分析结果一致。本文通过全面分析肺形侧耳低温胁迫时期基因表达情况,为进一步研究肺形侧耳低温胁迫相关重要基因的功能鉴定与信号通路调控机理提供基础。     

Determination of Pleurotus ostreatus, Pleurotus pulmonarius and Paxillus involutus toxicity over Artemia salina

We report the toxicity of ethanolic extracts in Pleurotus ostreatus, Pleurotus pulmonarius and Paxillus involutus over that obtained in Artemia salina. P. involutus showed the highest toxicity (LC50 = 94.4 microg/ml), similar to that detected using potassium dichromate pattern. P. pulmonarius and P. ostreatus did not show toxicity over A. salina in concentrations lower than 1,000 microg/ml.     

凤尾菇化学成分和药理活性研究进展

对凤尾菇的化学成分及药理活性的研究进展进行了综述。研究表明凤尾菇含有多糖类、氨基酸、蛋白质、三萜类、甾体类、脂肪酸类等多种化学成分,并具有免疫调节、抗肿瘤、抗氧化、抗衰老、抗菌抗炎、抗凝血等广泛的药理活性。目前对凤尾菇的化学成分研究较弱,主要集中在凤尾菇多糖药理活性方面,并取得了一定的进展。     

刺芹侧耳菌渣对肺形侧耳（秀珍菇）生长和营养成分的影响

以常规棉籽壳培养料为对照,用刺芹侧耳Pleurotus eryngii工厂化生产的废料（简称菌渣）代替部分棉籽壳进行格氏侧耳Pleurotus geesterani栽培试验,并测定格氏侧耳的营养成分。结果表明,供试培养料中菌丝长势均良好,随着菌渣替代比例的增加,菌丝满袋时间、原基出现时间和转潮期缩短,生长周期变短;菌渣代替比例增加,产量呈降低趋势,其中替代比例33%（1:2）和55%（1.2:1）时培养料的生物学效率与对照无显著差异,替代比例55%和78%（3.5:1）时培养料对第四潮和第五潮菇产量影响明显,显著低于对照;供试菌渣培养料栽培格氏侧耳的粗蛋白和总氨基酸含量略高于或显著高于对照,说明添加适量的菌渣栽培格氏侧耳可提高子实体的营养成分。     

自交选育秀珍菇新菌株“申秀1号”

【目的】对自交子代群体主要栽培和商品性状进行综合分析,并选育综合性状优良的秀珍菇新菌株。【方法】以秀珍菇商业菌株“3108”为亲本进行自交,综合分析其自交子代群体的菌丝生长速度、产量、菇型、抗杂、抗逆性和栽培周期等性状以选育新菌株,利用ISSR (Inter-sample sequence repeat)鉴定新菌株和亲本菌株之间遗传差异性。【结果】自交子代中,菌丝生长速度和产量分别有19.5%和8.9%的菌株优于亲本;子代群体中菌丝生长速度和子实体产量之间无相关性(R=0.102,P>0.05);37%的菌株栽培周期比亲本短;菇盖平展、厚且不易碎分别占53%、11%,菌柄直径和长度变化幅度分别为5?13 mm和21?75 mm;发生黄枯病,出现萎缩、死菇现象以及畸形分别占23%、19%、5%。【结论】通过对子代群体各性状综合分析,从中选育出生长快、产量高、菇盖厚且不易碎、柄粗长、抗杂和抗逆性强、栽培周期短等优良性状的秀珍菇新菌株“申秀1号”,前三潮菇平均单袋产量334 g,生物学效率高达74%,比亲本菌株增产7.1%,且种性稳定。经ISSR鉴定表明,该菌株具有自身特异性。     

Degradation of endosulfan during substrate preparation and cultivation of Pleurotus pulmonarius

Summary Endosulfan is an insecticide used on many vegetable crops. In mushroom cultivation, vegetable materials used as a growth substrate may contain residues of endosulfan that may accumulate in the final mushroom biomass. After preparing the substrate, it is subjected to pasteurization and/or composting and then inoculated with the desired fungus. The purpose of this research was to determine the rate and extent of endosulfan reduction from a grass substrate that was either composted or sterilized by autoclaving. In addition, the rate and extent of removal of endosulfan from substrate colonized with Pleurotus pulmonarius was determined. The degradation of 65 mg/kg endosulfan was analyzed on both, the substrate preparation and the culture of P. pulmonarius on the grass Digitaria decumbens. During composting in presence of Ca(OH)₂ for 120 h, the concentrations of α and β endosulfan were reduced by 61.4 and 49.5% respectively, significantly higher compared with the control (without Ca(OH)_2,) in which the reduction was 38.5%. After sterilization the concentration of α and β endosulfan was reduced by 84.8 and 87.5% respectively. After the colonization of substrate by P. pulmonarius (15 days after spawning) α and β endosulfan were reduced by 96% and at the end of cultivation (35 days after spawning) were reduced by 99%. When carpophores were analyzed, residues of α and β endosulfan were observed between 0.019–0.084 mg/kg. The results showed that α and β endosulfan were partially removed during the preparation of substrate and entirely eliminated during fungal colonization on the substrate.     

Microscopic observations of the early development of Pleurotus pulmonarius fruit bodies

From observations made by light microscopy, transmission electron microscopy, environmental-scanning and cryoscanning electron microscopy we conclude that the expansion of the young fruit body of Pleurotus pulmonarius involves considerable vacuolation of hyphae but no marked inflation of cell dimensions. There is evidence for an extensive extracellular matrix (ECM), the components of which must be under the control of the hyphae which the ECM surrounds. However the ECM in these fruit bodies is a dilute material. It is easily lost during specimen preparation and is evident only when certain techniques are used to preserve the fluid surface of the hyphae. Observations of the hyphal and fruit body structures with a range of conventional microscopic techniques are crucial to complement the information obtained through physiological and molecular studies for understanding the cellular changes that occur during mushroom development.     

肺形侧耳发酵培养基优化及多糖提取工艺

通过碳源、氮源单因素实验和正交实验,对野生肺形侧耳进行发酵培养基优化。选取浸提时间、浸提温度及液料比3个因素,以胞内粗多糖提取率为指标,采用正交实验设计确定菌丝体胞内多糖提取的最佳工艺。结果表明,适宜肺形侧耳深层发酵的培养基为蔗糖1.5%,麸皮5%,蛋白胨0.6%,KH₂PO₄ 0.15%,MgSO₄ 0.75%,VB₁ 0.01%。胞内多糖提取的最佳工艺为浸提时间2h,液料比50:1,浸提温度90℃,此条件下多糖提取率为34.35%。     

不同栽培基质对肺形侧耳营养成分及呈味物质的影响

以3种林木枝条废弃物(金银花、松木、杨木)作为栽培基质,对4株综合性状较优异的肺形侧耳菌株进行栽培,测定不同栽培基质的肺形侧耳子实体中粗蛋白、水解氨基酸、游离氨基酸、总糖及5'-核苷酸组成与含量,研究不同菌株、不同栽培基质下肺形侧耳子实体营养成分及呈味物质的含量,以柞木+棉籽壳传统栽培基质作为对照.结果 表明不同菌株之...     

A Novel Peroxidase from Fresh Fruiting Bodies of the Mushroom Pleurotus pulmonarius

International Journal of Peptide Research and Therapeutics - A novel peroxidase has been isolated from fresh fruiting bodies of the mushroom Pleurotus pulmonarius, with a purification protocol of...     

Toxicities of DDE on Wheat and Bioremediation of DDE by Fungus Pleurotus pulmonarius

An in vitro study was performed to determine the acute phytotoxicities and genotoxicity of DDE either spiked to soil or added to hydrophonic cultures on wheat Triticum aestivum. A 24-well plate was first used to determine toxicity on individual grains using conventional seed germination/seedling growth toxicity tests whereas a single cell electrophoresis system was applied to measure genotoxicity at single cell level for wheat. Hydrophonic cultures provide a simplified environment to screen for toxicities with high sensitivity. Inverse dose-response relationships were detected between exogenous DDE levels and one of the following parameters: seed germination, seedling growth, and genotoxicity. In contrast, soil reduced the stress on T. aestivum by lowering bioavailability leading to less DDE distributed in radicle and coleoptile, modulated growth, and enhanced tolerance. At all DDE doses spiked to soil including the reference safety level of 0.5 mg/kg, DNA breakage was detected in both radicle and coleoptile but their magnitudes did not correlate with the organ nor the soil DDE contents. Thus, although wheat is highly sensitive to the genotoxic effect of DDE, first demonstrated here, the seed germination test offers a simple quantitative measure of DDE's phytotoxicity in soil and hydrophonic cultures. This study also found that fungus Pleurotus pulmonarius, which secretes extracellular ligninolytic enzymes causing non-specific cleavage of lignin and organopollutants, remediated DDE spiked to soil. In 5 weeks, 78% of 10 mg/kg DDE was biodegraded, and the fungal-treated soil reduced acute toxicity on T. aestivum using the seed germination test.     

The effect of lignocellulose on lignocellulolytic activity of Pleurotus pulmonarius in submerged culture

Summary The possible effects of lignocellulose substrate on lignocellulolytic activity of the fungus Pleurotus pulmonarius was studied in submerged culture. This study included fungal growth rates, nitrogen and carbon consumption, and enzymatic activity rates, with and without added cotton-wheat straw (CWS) mixtures. Addition of CWS to the media caused increased consumption of glucose and NH _inf4 ^sup+ by the fungal mycelium, induced carboxymethylcellulase (CMC-ase), increased poly-B decolourization, and enhanced the activity of laccase tenfold, while -glucosidase activity was also enhanced: its first peak was higher and second peak earlier. Lignin peroxidase, however, was not detected. These results give some indication that the lignocellulolytic activity of P. pulmonarius in liquid culture is enhanced by the presence of lignocellulosic substrates such as CWS.Offprint requests to: D. Levanon     

Extract of Pleurotus pulmonarius suppresses liver cancer development and progression through inhibition of VEGF-induced PI3K/AKT signaling pathway

Liver cancer or hepatocellular carcinoma is one of the leading causes of cancer-related deaths. Conventional chemotherapies are limited by the development of drug resistance and various side effects. Because of its non-toxicity and potent biopharmacological activity, metabolites derived from mushrooms have received more attention in cancer therapy. Our previous studies have demonstrated the anticancer effects of polysaccharide-protein complexes derived from the Pleurotus mushrooms. The aim of this study was to investigate the underlying molecular mechanism of the anticancer activity of a hot water extract containing a polysaccharide-protein complex isolated from Pleurotus pulmonarius (PP) in liver cancer cells. Our results indicated that exposure of liver cancer cells to PP not only significantly reduced the in vitro cancer cell proliferation and invasion but also enhanced the drug-sensitivity to the chemotherapeutic drug Cisplatin. Both oral administration and intraperitoneal injection of PP significantly inhibited the tumor growth in xenograft BALB/c nude mice. PP triggered a marked suppression of the PI3K/AKT signaling pathway in liver cancer cells in vitro and in vivo, and overexpression of the constitutively active form of AKT, Myr-AKT, abrogated this effect and the inhibited proliferation and invasion by PP. Both western blot and ELISA results showed that PP-treated liver cancer cells had reduced expression and secretion of vascular endothelial growth factor (VEGF). Addition of recombinant human VEGF attenuated the inhibitory effects of PP on PI3K/AKT pathway and the cancer phenotypes. Our results demonstrated that PP suppressed the proliferation, invasion, and drug-resistance of liver cancer cells in vitro and in vivo, mediated by the inhibition of autocrine VEGF-induced PI3K/AKT signaling pathway. This study suggests the potential therapeutic implication of PP in the treatment of human liver cancer.     

Waste-reducing cultivation of Pleurotus ostreatus and Pleurotus pulmonarius on coffee pulp: changes in the production of some lignocellulolytic enzymes

Fruiting body production for one strain of Pleurotus ostreatus and three strains of P. pulmonarius was evaluated on coffee pulp pasteurized at 80 °C for 1 h. Based upon three harvests per strain, the single P. ostreatus line was found to display a 40-day culture cycle, whereas the three P. pulmonarius strains completed their cycles after more than 50 days of incubation. These time periods were notably shorter than those observed in previous studies using other growth substrates. Nevertheless, yields expressed as biological efficiencies were not significantly different among strains, fluctuating between 125 and 138%. Extracellular enzymatic activity was also monitored for P. ostreatus and P. pulmonarius (one strain only). To do this, samples of mycelium-bearing substrate were taken every 4 days throughout the incubation period. Care was taken to represent all developmental stages, including primordial and fruiting bodies. Samples were either lyophilized and then analysed or, in some cases, analysed immediately without lyophilization. Hydrolase activity (i.e. endoglucanase (CMC) and cellobiohydrolase (CBH)) was found to depend on developmental stage, showing peak production during fruiting body formation. On the other hand, oxidase activity-(i.e. laccase (LAC) and Mn-peroxidase (MnP)) was associated with phenol degradation. Nevertheless, in the case of oxidases developmental timing differences were also observed. Specifically, LAC activity was detected as early as 8 days after inoculation in non-lyophilized samples, whereas MnP appeared near the end of the incubation period. No LAC activity was observed in lyophilized samples. This study concludes that coffee pulp might be successfully employed in the cultivation of mushrooms, not only because important extracellular enzymes are produced by mushrooms when grown upon this substrate, but also because the abbreviated cultivation cycle associated with this medium favours commercial processes. Commercialization might be further improved if strains specifically adapted to this novel substrate are selected.