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.     

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...     

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。     

利用mRNA差异显示技术研究香菇发育相关基因

以香菇子实体不分化突变株和对照菌株L98411为材料,利用mRNA差异显示技术研究二者的基因表达差异,共分离到21条差异片段,其中12个分别与泛素、ATP合成酶、锌指蛋白、GTP结合蛋白、延伸因子g1线粒体前体、过氧化物酶、硫酯酶、类TrpB酶、2,4-二烯酰辅酶A还原酶、糖苷水解酶、热激蛋白、疏水蛋白等已知基因有较高的同源性,这表明香菇子实体分化发育是一个复杂的过程,涉及到胞内转运、转录调控、细胞分化、蛋白合成、各种代谢途径中的酶、抗逆反应等诸多途径的协同调控。     

Transcript profiling reveals novel marker genes involved in fruiting body formation in Tuber borchii

cDNA arrays were used to explore mechanisms controlling fruiting body development in the truffle Tuber borchii. Differences in gene expression were higher between reproductive and vegetative stage than between two stages of fruiting body maturation. We suggest hypotheses about the importance of various physiological processes during the development of fruiting bodies.     

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) from the mushroom Lentinula edodes: characterization of the cDNA and its expressed product

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA. The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S. pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs). The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5'GCAATGT3' (complementary; 5'ACATTGC3'). Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box. The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus. There exists a possible leucine zipper between the two phosphorylation sites. The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase. Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper. Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L. edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation.     

Cloning and characterization of two cellulase genes from Lentinula edodes

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma(32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degrees C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and DeltaclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.     

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。     

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     

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。     

Isolation and analysis of genes specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes by fluorescence differential display

Using fluorescence differential display, cDNAs specifically expressed at the primordial stage of fruiting body development were isolated from the basidiomycete, Flammulina velutipes. Seventy-five cDNAs were sequenced and compared with the amino-acid sequences of proteins in the database by BLASTX search. Significant similarity was found for 29 cDNAs coding for proteins with known function, GTP-binding protein, growth factor, ubiquitin-proteasome, cytochrome P450 and hydrophobin, all of which would be associated with fruiting body development. Seventeen cDNAs were not similar to proteins in the database and may represent unique genes that play specific roles in the process of fruiting in F. velutipes.     

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.