Identification and Functional Analysis of Pheromone and Receptor Genes in the B3 Mating Locus of Pleurotus eryngii

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.     

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。     

Veratryl alcohol stimulates fruiting body formation in the oyster mushroom, Pleurotus ostreatus

The oyster mushroom, Pleurotus ostreatus, cultivated in solid state on sugarcane bagasse-wheat bran (5:1) medium in the presence of veratryl alcohol resulted in an increased production of the fruiting body at earlier times compared to when the fungus was grown in the absence of veratryl alcohol. The results indicate a new physiological role for veratryl alcohol in stimulating fruiting body formation. Veratryl alcohol also stimulated laccase production during the mycelial growth stage. Evidence is also presented that laccases were involved in the physiological development of the fruiting body.     

Cultivation of different strains of king oyster mushroom (Pleurotus eryngii) on saw dust and rice straw in Bangladesh

Pleurotus eryngii is a popular mushroom due to its excellent consistency of cap and stem, culinary qualities and longer shelf life. In Bangladesh, where Pleurotus mushrooms are very popular, P. eryngii may take position among the consumers, but currently this mushroom is not cultivated in large scale there. In this study, 3 strains of P. eryngii such as Pe-1 (native to Bangladesh), Pe-2 (germplasm collected from China) and Pe-3 (germplasm collected from Japan) were cultivated on saw dust and rice straw and their growth and yield parameters were investigated. Pe-1 on saw dust showed the highest biological yield and efficiency (73.5%) than other strains. Also, the mycelium run rate and number of fruiting bodies were higher in Pe-1 than other two strains. The quality of mushroom strains was near about similar. On saw dust, the yield and efficiency were better than those cultivated on rice straw, however, on straw; the mushroom fruiting bodies were larger in size. This study shows the prospects of P. eryngii cultivation in Bangladesh and suggests further study in controlled environment for higher yield and production.     

漆酶同工酶基因在斑玉蕈生长发育过程中的表达分析

为了探明漆酶在斑玉蕈生长发育过程中的功能,对斑玉蕈转录测序预测的13个漆酶基因序列进行分析、鉴定和构建分子系统发育树;检测了不同生长发育时期漆酶的活性和漆酶基因表达水平。研究结果显示：13个基因片段中有10个是漆酶基因。不同的漆酶同工酶之间进化关系存在明显差异,大多数漆酶与木腐菌（金针菇Flammulina filiformis和侧耳属Pleurotus）进化关系较近。对斑玉蕈不同生长发育时期的酶活检测结果显示,从斑玉蕈的菌丝恢复期到钉头期,漆酶活性逐渐升高,而在子实体形成后期酶活逐渐降低。对培养40d、60d和80d的菌丝样品以及不同生长发育时期的样品进RT-qPCR检测,结果显示在菌丝营养生长时期,大多数漆酶基因在第40-60天表达量持续增加1-3倍,而在第60-80天时表达量出现降低的情况。而在生殖生长时期,大多数漆酶基因在转色期或者原基期相对表达量达到最大值,并在子实体期出现降低,这与漆酶活性的检测具有一致性。lcc3、lcc7、lcc8和lcc9在斑玉蕈生殖生长过程中相对表达量出现了10-100倍的上调。这说明从菌丝培养到菌丝扭结形成子实体和子实体发育的过程中,不同的漆酶可能发挥着不同的作用,表达量较高的漆酶基因可能对基质降解和子实体形成起主要作用。     

Evaluation of Burma Reed as Substrate for Production of Pleurotus eryngii

Burma reed (Neyraudia reynaudiana), a giant C₄ grass, was included in substrate at the rates of 0, 20, 40 and 66 % to partially or wholly substitute sawdust and cottonseed hulls to evaluate its suitability for Pleurotus eryngii cultivation. Inclusion of 20 and 40 % Burma reed did not significantly affect linear mycelial growth, dry matter loss, spawn run period and fructification, and achieved high fruiting body yields and biological efficiency of 336.67 g/bag, 67.33 % and 342.15 g/bag, 68.43 %, respectively, which were not significantly different from 350.08 g/bag to 70.02 % obtained from the control substrate. Enzyme assay revealed that on the mixed substrates laccase and manganese peroxidase activity were significantly enhanced, but cellulase was significantly reduced in the middle stage of incubation as compared with the control substrate. Even on Burma reed substrate without sawdust and cottonseed hulls, fruiting body yield (313.56 g/bag) and biological efficiency (62.71 %) were satisfactory, although significantly lower than that on the control substrate. Therefore, Burma reed was a promising potential substrate for P. eryngii production to largely substitute sawdust and cottonseed hulls.     

Molecular characterization and enzymatic activity of laccases in two Pleurotus spp. with different pathogenic behaviour

Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host.     

Relationship between fruiting body development and extracellular laccase production in the edible mushroom Flammulina velutipes

The biochemical mechanism underlying the development of fruiting bodies in Flammulina velutipes, an edible mushroom, was investigated using the YBLB colorimetric assay to distinguish between the normal strain (FVN-1) and the degenerate strain (FVD-1). In this assay, the color of the YBLB medium (blue-green) inoculated with FVN-1 exhibiting normal fruiting body development changed to yellow, while the color of the medium inoculated with FVD-1 changed to blue. In this study, we found that this color difference originated from extracellular laccase produced by FVN-1. Moreover, FVN-1 exhibited considerably higher extracellular laccase activity than FVD-1, under conditions facilitating fruiting body formation. Overall, these findings suggest that extracellular laccase is involved in the fruiting body development process in F. velutipes.     

Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation. Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.     

利用加权基因共表达网络分析方法挖掘香菇发育不同阶段的关键基因

香菇是世界产量第二大食用菌,栽培历史悠久。在木屑袋料栽培模式下,香菇发育可以分为菌丝生长期（G）、菌丝褐化期（B）、原基形成期（P）以及出菇期（FB）4个阶段。褐化期和原基形成期是香菇从营养生长期到生殖生长两个关键发育阶段,对香菇子实体产量和质量至关重要。本研究以3种不同栽培材料为重复,对香菇发育的前3个阶段进行了转录组分析。主成分分析和相似性分析表明,基因随着发育进程的推进,不同栽培基质样本的基因表达特征相似。以菌丝生长阶段的转录本为参照,通过基因差异表达分析,获得与菌丝褐化成熟和原基形成相关的基因,并对这些基因进行GO和KEGG功能富集分析;其次,对9个转录本数据进行加权基因共表达网络分析（WGCNA）,分别获得了与菌丝生长、褐化阶段及原基形成各阶段高度相关的黑色、蓝色及黄色基因模块,并利用网络节点分析获得了与菌丝褐化成熟和原基形成得到7个关键基因;最后,结合差异基因和基因模块分析,得到了菌丝生长阶段的17个重要基因、褐化阶段的167个重要基因以及原基形成阶段的67个重要基因。通过多分析手段结合为筛选候选基因提供了更为高效的方法。     

Dietary effect of Pleurotus eryngii on biochemical function and histology in hypercholesterolemic rats

This work was conducted to investigate diet supplement of king oyster mushroom fruiting bodies on biochemical and histological changes in hypercholesterolemic rats. Six-week old female Sprague–Dawley albino rats were divided into three groups of 10 rats each. The feeding of 5% powder of the fruiting bodies of Pleurotus eryngii to hypercholesterolemic rats reduced their plasma total cholesterol, triglyceride, low-density lipoprotein, total lipid, phospholipids, and LDL/HDL ratio by 24.05%, 46.33%, 62.50%, 24.63%, 19.22%, and 57.14%, respectively. Mushroom also significantly reduced body weight in hypercholesterolemic rats. However, it had no adverse effects on plasma albumin, total bilirubin, direct bilirubin, creatinine, blood urea nitrogen, uric acid, glucose, total protein, calcium, sodium, potassium, chloride, inorganic phosphate, magnesium, and enzyme profiles. Feeding mushroom increased total lipid and cholesterol excretion in feces. The plasma lipoprotein fraction, separated by agarose gel electrophoresis, indicated that P. eryngii significantly reduced plasma β and pre-β-lipoprotein, while increased α-lipoprotein. A histological study of hepatic cells by conventional hematoxylin–eosin and oil red O staining showed normal findings for mushroom-fed hypercholesterolemic rat. The present study suggests that 5% P. eryngii diet supplement provided health benefits by acting on the atherogenic lipid profile in hypercholesterolaemic rats. Therefore, king oyster mushroom could be recommended as a natural cholesterol lowering substance within the human diet.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

曲酸对斑玉蕈子实体形成过程中木质纤维素酶的影响研究

为了探究曲酸增加子实体产量的机制,首先考察了搔菌后外源添加曲酸对不同菌丝培养时间出菇的影响。研究发现当菌丝培养时间过短或者过长添加曲酸都得不到很好的增产效果,菌丝培养时间在60-80d之间增产效率最高,并且后熟期60d的增产效率大于80d的增产效率。进一步研究发现添加曲酸可以提高菌丝利用基质中木质纤维素的利用率。更深入地研究发现,基质中的漆酶和纤维素酶活性在斑玉蕈的不同发育时期受到曲酸调控。漆酶活性在最初的菌丝恢复期和转色期酶活性低于对照组,但是在原基期、钉头期和子实体期酶活性显著地高于对照组;纤维素酶活性在整个发育周期中曲酸组都高于对照组,在子实体发育后期酶活性被提高3.16倍。最后,从分子水平上分析了漆酶基因和纤维素酶基因的表达量,研究显示添加曲酸后漆酶基因和纤维素酶基因在不同程度上被上调,这个结果与酶活的结果相一致。这些结果说明外源添加曲酸通过提高生殖生长阶段的菌丝利用培养基质中的漆酶和纤维素酶活性,进而提高菌丝利用木质纤维素,为斑玉蕈子实体生长发育提供更多的能源,实现增加子实体产量的目的。     

Characterization, Molecular Cloning, and Differential Expression Analysis of Laccase Genes from the Edible Mushroom Lentinula edodes

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.