Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-beta-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

Isolation and Characterization of Differentially Expressed Genes in the Mycelium and Fruit Body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-β-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus

To characterize genes involved in fruit body development, two complementary DNA (cDNA) libraries were constructed from RNA isolated from liquid-cultured mycelia and fruit bodies of Pleurotus ostreatus. Using single-pass sequencing of cDNA clones, 952 and 1069 expressed sequence tags (ESTs) were generated from liquid-cultured mycelia and fruit body cDNA library, respectively. A BLASTX search revealed that 390 of the liquid-cultured mycelia ESTs (41%) and 531 of the fruit body ESTs (50%) showed significant similarity to protein sequences described in the nonredudant database (E values < or =1 x 10(-5)). When liquid-cultured mycelia and fruit body ESTs were compared by the SeqMan II program, among the total of 2021 ESTs, 1256 ESTs were unigenes, and 66 unigenes (5.3%) were commonly expressed during both stages. The functional catalogs of the ESTs were made by comparison with functionally identified Saccharomyces cerevisiae genes. Liquid-cultured mycelium ESTs were compared with fruit body ESTs and changes of the expressed genes during fruit body development were analyzed.     

Growth promotion of the edible fungus Pleurotus ostreatus by fluorescent pseudomonads

Bacteria were isolated from the mycelial surface of Pleurotus ostreatus and their role in fruiting body induction (fructification) of the edible mushroom P. ostreatus was investigated. Analysis of the bacterial community that colonized the mycelium showed that the species composition and numbers of culturable bacteria differed according to the developmental stage of P. ostreatus. In particular, the population size of fluorescent pseudomonads increased during fruiting body induction. An experiment showed that inoculation of pure cultures of the mycelium with strains of fluorescent Pseudomonas spp. isolated from the mycelial plane of commercially produced mushrooms promoted the formation of primordia and enhanced the development of the basidiome of P. ostreatus. Results of this research strongly suggest that inoculation of the mycelium with specific bacteria may have beneficial applications for mushroom production.     

Identification of Long Non-Coding RNAs and Their Target Genes from Mycelium and Primordium in Model Mushroom Schizophyllum commune

Schizophyllum commune has emerged as the most promising model mushroom to study developmental stages (mycelium, primordium), which are two primary processes of fruit body development. Long non-coding RNA (lncRNA) has been proved to participate in fruit development and sex differentiation in fungi. However, potential lncRNAs have not been identified in S. commune from mycelium to primordium developmental stages. In this study, lncRNA-seq was performed in S. commune and 61.56 Gb clean data were generated from mycelium and primordium developmental stages. Furthermore, 191 lncRNAs had been obtained and a total of 49 lncRNAs were classified as differently expressed lncRNAs. Additionally, 26 up-regulated differently expressed lncRNAs and 23 down-regulated between mycelium and primordia libraries were detected. Further, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differentially expressed lncRNAs target genes from the MAPK pathway, phosphatidylinositol signal, ubiquitin-mediated proteolysis, autophagy, and cell cycle. This study provides a new resource for further research on the relationship between lncRNA and two developmental stages (mycelium, primordium) in S. commune.     

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.     

Isolation of genes expressed during the developmental stages of the oyster mushroom, Pleurotus ostreatus, using expressed sequence tags

In order to isolate and identify the developmentally regulated genes during fruiting body development, cDNA libraries were constructed from eight developmental stages of the Oyster mushroom, Pleurotus ostreatus. From these libraries, 11 761 expressed sequence tags (PoESTs) were generated. Of these, 4060 different genes (PoUnigenes) were identified, representing 34.5% of the entire genome. Redundancy analysis of ESTs during the developmental stages identified eight, 13 and two genes that were specifically expressed in mycelia, fruiting body and basidiospore, respectively. RT-PCR was used to confirm the specific expression of nine genes which showed specific redundancy in fruiting body stages, four genes of which were expressed specifically in fruiting body stages as expected in redundancy analysis, and other genes were expressed abundantly in fruiting body stages. The EST database of P. ostreatus generated during this study provides a genetic and biochemical basis for future studies of the developmental stages of basidiomycetes.     

Analysis of gene expression in the vegetative and fructification phases of the white truffle, Tuber borchii Vittad., by mRNA differential display

The mRNA differential display technique was used to compare mRNA populations from fruit body and mycelium of a white truffle species in the attempt to identify and clone differentially expressed genes. The differential expression of five out of 30 amplicons was confirmed. One fragment (Tbm 56) corresponded to a part of the ribosomal genes. Three cDNA fragments (Tbf 12, Tbf 20, Tbf 21) were expressed only in the fructification phase, while the other cDNA (Tbf 55) was expressed strongly in fruit body and also detectable in the mycelium. These clones correspond to part of the single-copy genes in the Tuber borchii Vittad. genome.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

A genotype-specific pollen gene associated with self-incompatibility in Lycopersicon peruvianum

Self-incompatibility is a genetically controlled process used to prevent self-pollination. We report here the characterization of pollen cDNA clones of Lycopersicon peruvianum, and the identification of a genotype-specific pollen factor involved in self-incompatibility. To identify the latter, differential mRNA display RT-PCR was performed on pollen cDNAs from S12Sa and S11Sa genotypes. We isolated four cDNA fragments expressed preferentially in S12Sa pollen, and screened a cDNA library from S12Sa pollen with the four cDNA fragments to isolate the corresponding full length cDNAs. One of the four isolated cDNAs encoded part of an actin depolymerizing factor protein that we named LpADF. LpADF is highly homologous to actin depolymerizing factors of Arabidopsis thaliana, Lilium longiflorum, and Zea mays. RNA blot analysis revealed that LpADF is only expressed in mature pollen of the S12Sa genotype, and is therefore a candidate pollen factor in the gametophyte self-incompatibility system of L. peruvianum.     

荧光假单胞菌对食用菌的促生作用及其机理

从小麦根际分离到1株对食用菌有较强促生作用的荧光假单胞菌,命名为假单胞菌P2-10(Pseudomonas sp.P2-10)。该菌株与平菇天达300(Pleurotus ostreatus Td 300)、杏鲍菇杏6(Pleurotus eryngii X6)或鸡腿菇瑞7(Coprinus comatus R7)混合培养,可显著提高这些食用菌菌丝生长速度及诱导并促进子实体的形成和发育。假单胞菌P2-10对食用菌的促生作用来自其代谢产物,可能是通过其1-氨基环丙烷-1-羧酸(ACC)脱氨酶降解食用菌产生的ACC、减少食用菌乙烯的合成及乙烯对菌丝生长和子实体发育的抑制作用。     

DNA marking of some quantitative trait loci in the cultivated edible mushroom Pleurotus ostratus (Fr.) Kumm

Fungi of the genus Pleurotus, in particular, species Pleurotus ostreatus (common oyster mushroom) are among most cultivated fungi in the world. Due to intense rates of development of studies in this field, efficient breeding programs are highly required in the search for new P. ostreatus strains. The principal traits used worldwide for selecting strains are intensity of fruitbearing, fruit body cap color (for some consumptive markets), and mycelium growth rate. In this connection, the objective of this work was to study these quantitative traits and to find molecular markers, which could be employed to accomplish breeding programs. In general, we found 12 genomic loci (quantitative trait loci, QTLs) controlling mycelium growth rate of oyster and six QTLs responsible for the fruit body cap color. The genetic map of P. ostreatus was constructed, and all markers of quantitative traits found by us were located on this genetic map. The obtained linkage map can be a useful tool for the accomplishment of breeding programs to improve economically important traits of oyster mushroom.