基于质构仪质地多面分析法对香菇质地评价

香菇Lentinula edodes质地是影响其食味品质和货架期的一系列重要品质指标.本研究首次基于质构仪质地多面分析法(TPA),结合传统感官评级对ZX6(浙香6号)和Lsm9两个香菇亲本及其2个杂交菌株(L1*11和L4*6)子实体的不同部位(菌柄P1、菌盖顶部P2、菌盖中心P3和菌盖侧边P4)的7个质地指标和2个...     

Cataloging and profiling genes expressed in Lentinula edodes fruiting body by massive cDNA pyrosequencing and LongSAGE

This study investigated the molecular mechanism of the fruiting body development and sporulation in the cap of the Shiitake mushroom, Lentinula edodes. Although there has been much research into L. edodes, there remain significant gaps in our knowledge of how the species reproduces. In order to provide molecular resources and to understand the molecular mechanism of the fruiting body development in basidiomycete comprehensively, we searched for the genes which are important for fruiting body development and sporulation in the cap of mature fruiting body of L. edodes by using the whole-genome approach. Massive cDNA pyrosequencing was used to generate >7000 sequence contigs from mature fruiting bodies. We used Gene Ontology to categorize the contigs to form the catalog of genes expressed at the stage of the mature fruiting body. We also assigned the contigs into the KEGG pathways. The catalog of expressed genes indicates that the mature fruiting bodies (1) sense the external environment, (2) transmit signals to express genes through regulatory systems, (3) produce many proteins, (4) degrade unwanted proteins, (5) perform extensive biosynthesis, (6) generate energy, (7) regulate the internal environment, (8) transport molecules, (9) carry out cell division, and (10) differentiate and develop. After establishing the catalog of expressed genes in L. edodes, we used the LongSAGE approach to analyze the expression levels of genes found in mature fruiting bodies before (FB) and after (FBS) spores appeared. Gene-expression patterns according to GO categories were similar in these two stages. We have also successfully identified genes differentially expressed in FB and FBS. Fold-changes in expression levels of selected genes based on LongSAGE tag counts were similar to those obtained by real-time RT-PCR. The consistency between real-time RT-PCR and LongSAGE results indicates reliability of the LongSAGE results. Overall, this study provides valuable information on the fruiting processes of L. edodes through a combination of massive cDNA pyrosequencing and LongSAGE sequencing, and the knowledge thereby obtained may provide insight into the improvement of the yield of commercially grown Shiitake mushrooms.     

球状香菇菌株的氨基酸特征分析及蛋白质品质评价

比较分析香菇球状菌株与正常菌株的形态特征、氨基酸特征和蛋白质品质,并基于现行国际氨基酸模式谱,采用蛋白质的氨基酸评分（amino acid score,AAS）、氨基酸比值系数分（ratio coefficient of amino acid,SRC）、IOM（Institute of Medicine）模式评分、化学评分（chemical score,CS）、必需氨基酸指数（essential amino acid index,EAAI）以及蛋白质校正氨基酸计分（protein digestibility corrected amino acids score,PDCAAS）多种指标进行评价。结果表明：与正常菌株相比,球状菌株形态上没有菌褶和菌柄的分化,同时营养成分也发生了变化,就粗蛋白含量而言,球状菌株为正常菌株的1.37倍（分别为32.32%和23.60%）;就平均总氨基酸含量而言,球状菌株（209.58mg/g）是正常菌株（163.10mg/g）的1.28倍。这些球状菌株的特征可作为新品种的培育材料进一步被利用。     

Developmental regulator Le.CDC5 of the mushroom Lentinula edodes: analyses of its amount in each of the stages of fruiting-body formation and its distribution in parts of the fruiting bodies

Immunoblot analysis of Le.CDC5 (842 amino acid residues), the expressed product of the cDNA of Le.cdc5 gene that has been previously reported to be most actively transcribed in primordia and small immature fruiting bodies of the basidiomycete Lentinula edodes, showed that the primordia, immature fruiting bodies and mature fruiting bodies contain similar amounts of Le.CDC5 protein. This indicates that the Le.CDC5 protein molecules synthesized in the beginning and early stage of fruiting-body formation remains in mycelial tissues even after small immature fruiting bodies developed and matured. Immunohistochemical analysis showed that Le.CDC5 is present everywhere in the mycelial tissues of immature fruiting body, but prehymenophore, the border between pileus and stipe, and the bottom of stipe seem likely to contain larger amounts of Le.CDC5. Within the hymenophore of mature fruiting body, the hymenium (in/on which a large number of basidia and basidiospores are formed) contains the Le.CDC5 most exclusively.     

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.     

Catalytic Reaction of Basidiomycete Lentinula edodes Cytochrome P450, Le.CYP1 Enzyme Produced in Yeast

The basidiomycete Lentinula edodes (Le.) cytochrome P450, Le.CYP1 was functionally expressed in Saccharomyces cerevisiae. The microsomal fraction containing Le.CYP1 was prepared from the recombinant yeast and the Le.CYP1 was analyzed. The 7-ethoxycoumarin and benzo(a)pyrene were found to be the substrates of Le.CYP1 enzyme. Le.CYP1 converted 7-ethoxycoumarin to 7-hydroxycoumarin.     

Preparation of Lentinula edodes polysaccharide-calcium complex and its immunoactivity

Polysaccharide is a major bioactive component of mushrooms. In this study, for the first time, starting from a new Lentinula edodes polysaccharide L2, we prepared a novel L2–calcium complex and the process was optimized. Scanning electron microscopy and Fourier Transform infrared spectrometry were used for characterization. The immunostimulating activities of L2 and L2–calcium complex were measured by enhancing the production of two cytokines TNF-α and IL-6 in RAW264.7 cells. While L2–calcium complex significantly stimulates the secretions of TNF-α and IL-6 compared with the control, complex with calcium ion decreased the secretion of them. These facts indicate that calcium ion can modulate immune stimulating activity of Lentinula edodes polysaccharide L2.     

香菇中麦角钙化甾醇含量的测定及其在人体内的传输机制

在阳光照射和自然阴干两种干燥方式下,测定了香菇中麦角钙化甾醇的含量。在不同实验条件下,测定了麦角甾醇转化为麦角钙化甾醇的转化率。在模拟生理条件下,通过荧光光谱、同步荧光光谱、三维荧光光谱、位点竞争实验、紫外可见吸收光谱和分子对接方法对麦角钙化甾醇与人血清白蛋白相互作用过程中的机制和构象变化进行了系统研究,从而揭示了麦角钙化甾醇在体内的传输机制。结果表明,阳光照射能够提高香菇中麦角钙化甾醇含量。麦角甾醇在溶液状态下经过紫外光照射4h,麦角甾醇转化为麦角钙化甾醇的转化率为21%。荧光光谱表明麦角钙化甾醇通过静态猝灭机制猝灭人血清白蛋白的内源荧光。在288K时有利于麦角钙化甾醇与人血清白蛋白的结合,在较高温度下麦角钙化甾醇不能与人血清白蛋白结合。热力学参数分析和分子对接结果表明,疏水作用、氢键和范德华力是结合过程中的主要作用力。位点竞争实验和同步荧光光谱表明位点I是麦角钙化甾醇和人血清白蛋白相互作用的主要结合位点。结合过程中能够发生能量转移,结合距离是3.46nm。结合过程轻微地改变人血清白蛋白的结构和微环境。     

香菇不同发育阶段子实体多糖的理化性质及体外免疫活性研究

香菇多糖是从香菇菌丝体或子实体中提取纯化得到的高分子葡聚糖,作为香菇主要的生物活性物质,具有提高免疫力、抗病毒、抗氧化和抗真菌等生物活性。本研究对幼菇期、菌褶期、采收期、成熟期和开伞期5个不同发育阶段香菇粗多糖含量、理化性质和体外免疫活性进行比较分析。结果表明,其粗多糖含量、理化性质和体外免疫活性具有明显差异。粗多糖得率呈现先增加后降低的趋势,菌褶期得率最高;粗多糖含量也呈现先增加后降低的趋势,成熟期含量最高;多糖的分子量因发育期不同而有较大差异,发育后期所得粗多糖分子量千万级以上组分的比例增加,分子量百万级和十万级的多糖组分比例降低;不同发育阶段香菇子实体多糖的单糖组成均包含果糖、半乳糖、葡萄糖和甘露糖4种单糖,且都以葡萄糖为主;5个阶段的多糖组分均有较好的体外免疫活性,其中幼菇期粗多糖在浓度500μg/mL时表现出最高的体外免疫活性。本研究探讨了香菇不同发育阶段子实体多糖的理化性质及体外免疫活性,为香菇采收期的确定及香菇多糖的制备与利用提供理论依据。     

Intracellular levels of cyclic AMP and adenylate cyclase activity during mycelial development in fruiting body formation in Lentinus edodes

Abstract Streptococcal promoters were shown to fulfil primary structure requirements for the expression of heterologous genes in Pseudomonas putida KT2440. The identity of the gene products, streptokinase and human interferon-α1, as synthesized by KT2440, was confirmed by their biological, antigenic and/or physical properties. Although designed as secretion vectors with streptococcal signal sequences, the recombinant plasmids failed to mediate the complete export of either protein into the periplasmic space of KT2440.     

DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

Influence of contamination by Penicillium brevicompactum and Trichoderma harzianum during Lentinula edodes spawn run on fruiting in sawdust-based substrates

 Substrates with two kinds of supplements, raw and deoiled rice bran, were artificially infected with Penicillium brevicompactum or Trichoderma harzianum on days 0, 26, 61, and 90 after inoculation with Lentinula edodes. With P. brevicompactum infection, there was no significant difference in the yield and size of the fruit-bodies among either infected and uninfected substrates, raw and deoiled rice bran substrates, or days when the substrates were infected. However, the irregularly shaped fruit-bodies, which were commercially of low value, yielded greatly on raw rice bran substrates infected on days 0 and 26, whereas the substrates infected with T. harzianum on any day were covered with conidia and fatally damaged. Received: August 29, 2001 / Accepted: February 15, 2002