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Abstract Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H2S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H2S concentration. Then, we observed that the carbon repressor CreA inhibited H2S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H2S biosynthesis, at “CTGGGG”. In our study, we reported for the first time that H2S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H2S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H2S signals.

Hydrogen sulfide (H2S) has recently been recognized as an important gaseous transmitter with multiple physiological effects in various species. Previous studies have shown that H2S alleviated heat-induced ganoderic acids (GAs) biosynthesis, an important quality index of Ganoderma lucidum. However, a comprehensive understanding of the physiological effects and molecular mechanisms of H2S in G. lucidum remains unexplored. In this study, we found that heat treatment reduced the mitochondrial membrane potential (MMP) and mitochondrial DNA copy number (mtDNAcn) in G. lucidum. Increasing the intracellular H2S concentration through pharmacological and genetic means increased the MMP level, mtDNAcn, oxygen consumption rate level and ATP content under heat treatment, suggesting a role for H2S in mitigating heat-caused mitochondrial damage in G. lucidum. Further results indicated that H2S activates sulfide-quinone oxidoreductase (SQR) and complex III (Com III), thereby maintaining mitochondrial homeostasis under heat stress in G. lucidum. Moreover, SQR also mediated the negative regulation of H2S to GAs biosynthesis under heat stress. Furthermore, SQR might be persulfidated under heat stress in G. lucidum. Thus, our study reveals a novel physiological function and molecular mechanism of H2S signalling under heat stress in G. lucidum with broad implications for research on the environmental response of microorganisms.

Abstract Background Edible mushrooms are delicious in flavour and rich in high-quality protein and amino acids required by humans. A transcription factor, general control nonderepressible 4 (GCN4), can regulate the expression of genes involved in amino acid metabolism in yeast and mammals. A previous study revealed that GCN4 plays a pivotal role in nitrogen utilization and growth in Ganoderma lucidum. However, its regulation is nearly unknown in mushrooms. Results In this study, we found that the amino acid contents reached 120.51 mg per gram of mycelia in the WT strain under 60 mM asparagine (Asn) conditions, but decreased by 62.96% under 3 mM Asn conditions. Second, silencing of gcn4 resulted in a 54.2% decrease in amino acid contents under 60 mM Asn, especially for the essential and monosodium glutamate-like flavour amino acids. However, these effects were more pronounced under 3 mM Asn. Third, silencing of gcn4 markedly inhibited the expression of amino acid biosynthesis and transport genes. In addition, GCN4 enhanced the tricarboxylic acid cycle (TCA) and glycolytic pathway and inhibited the activity of target of rapamycin complex 1 (TORC1), thus being beneficial for maintaining amino acid homeostasis. Conclusion This study confirmed that GCN4 contributes to maintaining the amino acid contents in mushrooms under low concentrations of nitrogen. In conclusion, our study provides a research basis for GCN4 to regulate amino acid synthesis and improve the nutrient contents of edible mushrooms.

ABSTRACT In the current study, the silencing of protein arginine methyltransferase 5 (PRMT5) decreased the polysaccharide content of Ganoderma lucidum compared to that of the wild-type (WT) strain. Furthermore, RNA-seq screening showed that the glycan degradation process-related gene thaumatin-like protein (TLP) was alternatively spliced in WT strains via retained introns, leading to the production of a longer TLP1 isoform and a shorter TLP2 isoform; however, only the TLP2 isoform was observed in PRMT5i strains. Experiments examining the polysaccharide content of the TLP silencing, TLP1 overexpression (OE-TLP1), and TLP2 overexpression (OE-TLP2) transformants revealed that TLP2 plays a more important role than TLP1 in polysaccharide degradation. Through a combination of yeast two-hybrid, bimolecular fluorescence complementation and surface plasmon resonance assays, we found that TLP2 directly physically interacted with phosphoglucose isomerase (PGI), a key enzyme in polysaccharide synthesis, and thereby increased PGI activity. However, TLP1 failed to interact with PGI, and PGI activity was not affected. Further inspection showed that the polysaccharide content was decreased in the OE-TLP2 strains but not significantly changed in the OE-TLP1 strains compared with that in the WT strains. In addition, the polysaccharide content of the PRMT5-TLP-cosilenced strains was not significantly different from that of the WT strains. These results demonstrate that PRMT5 modulates TLP processing of pre-mRNA transcripts and thereby decreases the polysaccharide content. IMPORTANCE PRMT5 contributes to secondary metabolite biosynthesis in Ganoderma lucidum. However, the mechanism through which PRMT5 regulates the biosynthesis of secondary metabolites remains unclear. In the current study, PRMT5 silencing led to a significant decrease in the biosynthesis of polysaccharides from G. lucidum through the action of the alternative splicing of TLP. A shorter TLP2 isoform can directly bind to PGI and regulated polysaccharide biosynthesis. These results suggest that PRMT5 enhances PGI activity by regulating TLP binding to PGI. The results of the current study reveal a novel target gene for PRMT5-mediated alternative splicing and provide a reference for the identification of PRMT5 regulatory target genes.

ABSTRACT Water stress affects both the growth and development of filamentous fungi; however, the mechanisms underlying their response to water stress remain unclear. In this study, water stress was found to increase intracellular reactive oxygen species (ROS) level, ganoderic acid (GA) content, and NADPH oxidase (NOX) activity of Ganoderma lucidum by 148.45%, 75.32%, and 161.61%, respectively. Water stress induced the expression of the G. lucidum aquaporin (GlAQP) gene, which facilitated water transfer for microbial growth. Compared to wild type (WT), exposure to water stress increased growth inhibition rate, ROS level, and GA content of GlAQP-silenced strains by 37 to 41%, 36 to 38%, and 25%, respectively. Furthermore, at the early stage of fermentation in GlAQP-silenced strains, water stress resulted in 16 to 17% and 9 to 10% lower ROS level and GA content compared to WT, respectively. However, in GlAQP-overexpressing strains, ROS level and GA content were 22 to 24% and 12 to 13% higher than in WT, respectively. In GlAQP-silenced strains, water stress at the late stage resulted in 35 to 37% and 29 to 30% higher ROS level and GA content, respectively, while in GlAQP-overexpressing strains, levels were 16 to 17% and 9% lower than WT, respectively. Cross talk between GlAQP and NOX positively regulated the GA biosynthesis of G. lucidum via ROS under water stress at the early stage but this regulation became negative at the late stage. This study deepens the understanding of fungal signaling transduction under water stress and provides a reference for analyzing environmental factors that influence the regulation of the fungal secondary metabolism. IMPORTANCE Ganoderma lucidum is an advanced basidiomycete that produces medicinally active secondary metabolites (especially ganoderic acid [GA]) with high commercial value. Water stress imposes an important environmental challenge to G. lucidum. The mechanism of GA biosynthesis under water stress and the role of G. lucidum aquaporin (GlAQP) during its biosynthesis remain unclear. Moreover, the effect of the relationship between GlAQP and NADPH oxidase (NOX) on the level of reactive oxygen species and GA production under water stress is unknown. This study provides information on the biological response mechanism of G. lucidum to water stress. A new theory on the cell signaling cascade of G. lucidum tolerance to water stress is provided that also incorporates the biosynthesis of secondary metabolites involved in NOX and GlAQP.

Liu et al. identify catalase as a target of S-nitrosylation by GSNOR and the specific sites of S-nitrosylation critical for its role in regulating secondary metabolite production in Ganoderma lucidum under heat stress. This study suggests that GSNOR regulates other metabolic pathways in microorganisms through S-nitrosylation of target proteins in response to environmental changes.

ABSTRACT Putrescine (Put) has been shown to play an important regulatory role in cell growth in organisms. As the primary center regulating the homeostasis of polyamine (PA) content, ornithine decarboxylase antizyme (AZ) can regulate PA content through feedback. Nevertheless, the regulatory mechanism of Put is poorly understood in fungi. Here, our analysis showed that GlAZ had a modulate effect on intracellular Put content by interacting with ornithine decarboxylase (ODC) proteins and reducing its intracellular protein levels. In addition, GlAZ upregulated the metabolic pathway of ganoderic acid (GA) biosynthesis in Ganoderma lucidum by modulating the intracellular Put content. However, a target of rapamycin (TOR) was found to promote the accumulation of intracellular Put after the GlTOR inhibitor Rap was added exogenously, and unbiased analyses demonstrated that GlTOR may promote Put production through its inhibitory effect on the level of GlAZ protein in GlTOR-GlAZ-cosilenced strains. The effect of TOR on fungal secondary metabolism was further explored, and the content of GA in the GlTOR-silenced strain after the exogenous addition of the inhibitor Rap was significantly increased compared with that in the untreated wild-type (WT) strain. Silencing of TOR in the GlTOR-silenced strains caused an increase in GA content, which returned to the WT state after replenishing Put. Moreover, the content of GA in GlTOR-GlAZ-cosilenced strains was also not different from that in the WT strain. Consequently, these results strongly indicate that GlTOR affects G. lucidum GA biosynthesis via GlAZ. IMPORTANCE Research on antizyme (AZ) in fungi has focused on the mechanism by which AZ inhibits ornithine decarboxylase (ODC). Moreover, there are existing reports on the regulation of AZ protein translation by TOR. However, little is known about the mechanisms that influence AZ in fungal secondary metabolism. Here, both intracellular Put content and GA biosynthesis in G. lucidum were shown to be regulated through protein interactions between GlAZ and GlODC. Furthermore, exploration of upstream regulators of GlAZ suggested that GlAZ was regulated by the upstream protein GlTOR, which affected intracellular Put levels and ganoderic acid (GA) biosynthesis. The results of our work contribute to the understanding of the upstream regulation of Put and provide new insights into PA regulatory systems and secondary metabolism in fungi.

Flammulina filiformis, the most produced edible mushroom species in China, is rich in lysine. Further enhancing its lysine biosynthesis is vital for improving its quality in industrialized cultivation. Citric acid induction significantly increases both the biomass and growth rate of F. filiformis hyphae, as well as the lysine content. The genes encoding enzymes in the lysine biosynthesis pathway were detected under the optimal induction, revealing that the expression levels of hcs, hac, and hah were 2.67, 1.97, and 1.90 times greater, respectively, relative to the control, whereas no significant difference was seen for hdh, aat, sr, and shd, and the expression of aar decreased. Furthermore, the transcriptional levels of Ampk, GCN2, GCN4, and TOR were found significantly upregulated, with the most upregulated, Ampk, reaching a level 42.68 times greater than that of the control, while the phosphorylation of AMPK rose by nearly 54%. In AMPK-silencing strains under the optimal induction, however, the phosphorylation increment dropped to about 16% and the lysine content remained at the same level as in the WT. Thus, AMPK is presented as the critical intermediary in citric acid’s regulation of lysine biosynthesis in F. filiformis.

The cultivation of crops in greenhouses is well established in China. However, the greenhouse climate is complex, rendering it difficult to analyze the greenhouse load and control the energy supply system. It is important to ensure the optimal design of greenhouse operation to enable optimal crop growth and maintain low operation costs for improving the greenhouse crop production efficiency and economic benefits. To reduce the energy consumption , we propose a new integrated energy supply system. This system uses a combined cooling, heating and power system, and an air source heat pump. A two-stage integrated optimization model of the integrated energy supply system was built, with “minimum average daily economic cost” and “maximum CO2 emission reduction rate (ERR)” as the objectives in the first stage. The characteristics of the countryside were taken into consideration for optimizing the capacity of the combined cooling, heating and power supply, energy storage and air source heat pump. In the second stage, the objectives were “maximum annual operating saving rate (OSR)”, “maximum CO2 emission reduction rate”, and “maximum primary energy saving rate (PESR)”. The capacity of the equipment designed in the first stage is used as the constraint to optimize the operating output of the combined cooling, heating and power supply, air source heat pump. Finally, a greenhouse is used as a prototype to carry out simulation calculations for two typical days in summer and winter to verify the effectiveness of this method. The simulation results showed that after the first stage of optimization, the average daily operating cost was significantly lower and the CO2 emission rate was reduced by 49.53%. After the second stage, annual operating saving rate, CO2 emission reduction rate, and primary energy saving rate were 39.51, 48.88, and 27.57% for a typical summer day and 52.96, 50.18, and 50.13% for a typical winter day, respectively. Therefore, compared to the existing energy supply systems, the integrated energy supply system and the integrated optimization design of the system could effectively avoid energy wastage, significantly improve the degree of match between the system design and operation, reduce operating cost, and reduce CO2 emission.

Abstract Circularly polarized light (CPL) detection has emerged as a key technology for various optoelectronics. Chiral hybrid perovskites (CHPs) that combine CPL‐sensitive absorption induced by chiral organic ligands and superior photoelectric properties of perovskites are promising candidates for direct CPL detection. To date, most of the CHP detectors are made up of polycrystalline thin‐film, which results in a rather limited discrimination of CPL due to the existence of redundant impurities and intrinsic defect states originating from rapid crystallization process. Here, it is developed a direct CPL detector with high photocurrent and polarization selectivity based on low‐defect CHP single‐crystal nanowire arrays. Large‐scale CHP nanowires are obtained through a micropillar template‐assisted capillary‐bridge rise approach. Thanks to the high crystallinity and ordered crystallographic alignment of these arrays, a CPL photodetector with high light on/off ratio of 1.8 × 104, excellent responsivity of 1.4 A W−1, and an outstanding anisotropy factor of 0.24 for photocurrent has been achieved. These results would provide useful enlightenment for direct CPL detection in high‐performance chiral optoelectronics.

Carbon monoxide (CO), a product of organic oxidation processes, arises in vivo principally from the enzymatic reaction of heme oxygenase (HO, transcription gene named HMX1). HO/CO has been found to exert many salutary effects in multiple biological processes, including the stress response. However, whether HO/CO is involved in the regulation of the heat-stress (HS) response of Ganoderma lucidum (G. lucidum) is still poorly understood. In this paper, we reported that under heat stress, the HMX1 transcription level, HO enzyme activity, and CO content increased by 5.2-fold, 6.5-fold and 2-fold, respectively. HMX1 silenced strains showed a 12% increase in ganoderic acid (GA) content under HS as analyzed by HPLC. Furthermore, according to Western blot analysis of the protein phosphorylation levels, HMX1 attenuated the increase in phosphorylation levels of slt2, but the phosphorylation levels were prolonged over a 3 h HS time period. The chitin and glucan content in HMX1 silenced strains increased by 108% and 75%, respectively. In summary, these findings showed that the HO/CO system responds to heat stress and then regulates the HS-induced GA biosynthesis and the cell-wall integrity mediated by the Slt-MAPK phosphorylation level in G. lucidum.

Lysine content is considered an important indicator of the quality of Flammulina filiformis. In this study, chitosan was used to improve lysine content of F. filiformis. Optimal design conditions were obtained using central combination design (CCD): treatment concentration was 14.61 μg/mL, treatment time was 52.90 h, and the theoretical value of lysine content was 30.95 mg/g. We used Basic Local Alignment Search Tool Protein (BLASTP) to search the F. filiformis genome database using known AATs in the NCBI database. There were 11 members of AAT in F. filiformis. The expression levels of AAT3 and AAT4 genes increased significantly with chitosan treatment. Subsequently, AAT3 and AAT4 silencing strains were constructed using RNAi technology. The lysine content of the wild-type (WT) strain treated with chitosan increased by 26.41%. Compared with the chitosan-induced WT strain, chitosan-induced lysine content decreased by approximately 24.87% in the AAT3 silencing strain, and chitosan-induced lysine content in the AAT4 silencing strain increased by approximately 13.55%. The results indicate that AAT3 and AAT4 are involved in the regulation of the biosynthesis of lysine induced by chitosan in F. filiformis. AAT3 may participate in the absorption of lysine, and AAT4 may be involved in the excretion of lysine with chitosan treatment.

Flammulina filiformis, previously known as Asian Flammulina velutipes, is one of the most commercially important edible fungi, with nutritional value and medicinal properties worldwide. However, precision genome editing using CRISPR/Cas9, which is a revolutionary technology and provides a powerful tool for molecular breeding, has not been established in F. filiformis. Here, plasmids harboring expression cassettes of Basidiomycete codon-optimized Cas9 and dual sgRNAs targeting pyrG under the control of the gpd promoter and FfU6 promoter, respectively, were delivered into protoplasts of F. filiformis Dan3 strain through PEG-mediated transformation. The results showed that an efficient native U6 promoter of F. filiformis was identified, and ultimately several pyrG mutants exhibiting 5-fluorooric acid (5-FOA) resistance were obtained. Additionally, diagnostic PCR followed by Sanger sequencing revealed that fragment deletion between the two sgRNA target sites or small insertions and deletions (indels) were introduced in these pyrG mutants through the nonhomologous end joining (NHEJ) pathway, resulting in heritable changes in genomic information. Taken together, this is the first report in which a successful CRISPR/Cas9 genome-editing system based on dual sgRNAs was established in F. filiformis, which broadens the application of this advanced tool in Basidiomycetes.

Ganoderma lucidum is a white-rot fungus that produces a range of lignocellulolytic enzymes to decompose lignin and cellulose. The mitogen-activated protein kinase (MAPK) pathway has been implicated in xylanases and cellulases production. As the downstream transcription factor of Slt2-MAPK, the function of Swi6 in G. lucidum has not been fully studied. In this study, the transcription factor GlSwi6 in G. lucidum was characterized and shown to significantly positively regulate cellulases and xylanases production. Knockdown of the GlSwi6 gene decreased the activities of cellulases and xylanases by approximately 31%~38% and 54%~60% compared with those of the wild-type (WT) strain, respectively. Besides, GlSwi6 can be alternatively spliced into two isoforms, GlSwi6A and GlSwi6B, and overexpression of GlSwi6B increased the activities of cellulase and xylanase by approximately 50% and 60%, respectively. Further study indicates that the existence of GlSwi6B significantly increased the concentration of cytosolic Ca2+. Our study indicated that GlSwi6 promotes the activities of cellulase and xylanase by regulating the Ca2+ signaling. These results connected the GlSwi6 and Ca2+ signaling in the regulation of cellulose degradation, and provide an insight for further improvement of cellulase or xylanase activities in G. lucidum as well as other fungi.

Coal gangue-based cemented backfill material (CGCBM) is developed for backfilling the goaf in coal mines. As fresh CGCBM slurry is generally transported into underground openings through a pipeline, and after hardening, it plays the role of supporting the overlying strata. The fluidity, stability, and mechanical (compressive strength) of CGCBM become the most important properties. Adding water-reducing agents (WRAs) is considered to improve the fluidity, stability, and mechanical properties of CGCBM, but there is a risk of increased bleeding. So, two types of WRA (naphthalene series (WRA1) and poly carboxylic acid (WRA2)) are used at different contents (1.0%-2% for WRA1, 0.2%–0.6% for WRA2) by mass of binder. Slump, slump flow, yield stress, and plastic viscosity test are used to evaluate the fluidity properties of CGCBM after adding WRA. Bleeding rate test is used to evaluate the stability of CGCBM after adding WRA. Compressive strength is the most important factor in measuring the mechanical properties. SEM and XRD tests are used to analyse the mechanism of strength change. Results show that the slump, slump flow, and plastic viscosity increase after adding WRA, which reduces the yield stress and improves the fluidity. The bleeding rate increases with the increase of WRA content, leading to a decrease in stability. Adding WRA increases the compressive strength, and it increases first and then decreases with the increase of the content at the later stage. Considering the effects of WRA on the fluidity, stability, and compressive strength properties of CGCBM, the reasonable content of WRA1 and WRA2 is 1.5% and 0.4%, respectively. The research results provide guidance for the design and preparation of CGCBM with favourable performance in practical production.

Ganoderma lucidum has high commercial value because it produces many active compounds, such as ganoderic acids (GAs). Salicylic acid (SA) was previously reported to induce the biosynthesis of GA in G. lucidum. In this study, we found that SA induces GA biosynthesis by increasing ROS production, and further research found that NADPH oxidase-silenced strains exhibited a partial reduction in the response to SA, resulting in the induction of increased ROS production. Furthermore, the localization of ROS shows that mitochondria are sources of ROS production in response to SA treatment. An additional analysis focused on the relationship between SA-induced ROS production and mitochondrial functions, and the results showed that inhibitors of mitochondrial complexes I and II exert approximately 40–50% superimposed inhibitory effects on the respiration rate and H2O2 content when co-administered with SA. However, no obvious superimposed inhibition effects were observed in the sample co-treated with mitochondrial complex III inhibitor and SA, implying that the inhibitor of mitochondrial complex III and SA might act on the same site in mitochondria. Additional experiments revealed that complex III activity was decreased 51%, 62% and 75% after treatment with 100, 200, and 400 µM SA, respectively. Our results highlight the finding that SA inhibits mitochondrial complex III activity to increase ROS generation. In addition, inhibition of mitochondrial complex III caused ROS accumulation, which plays an essential role in SA-mediated GA biosynthesis in G. lucidum. This conclusion was also demonstrated in complex III-silenced strains. To the best of our knowledge, this study provides the first demonstration that SA inhibits complex III activity to increase the ROS levels and thereby regulate secondary metabolite biosynthesis. Keywords: Salicylic acid, Reactive oxygen species, Mitochondrial complex III, Ganoderic acids

Anti-pollution flashover of insulator is important for power systems. In recent years, haze-fog weather occurs frequently, which makes discharge occurs easily on the insulator surface and accelerates insulation aging of insulator. In order to study the influence of haze-fog on the surface discharge of room temperature vulcanized silicone rubber, an artificial haze-fog lab was established. Based on four consecutive years of insulator contamination accumulation and atmospheric sampling in haze-fog environment, the contamination configuration appropriate for RTV-coated surface discharge test under simulation environment of haze-fog was put forward. ANSYS Maxwell was used to analyze the influence of room temperature vulcanized silicone rubber surface attachments on electric field distribution. The changes of droplet on the polluted room temperature vulcanized silicone rubber surface and the corresponding surface flashover voltage under alternating current (AC), direct current (DC) positive polar (+), and DC negative polar (−) power source were recorded by a high speed camera. The results are as follows: The main ion components from haze-fog atmospheric particles are NO3−, SO42−, NH4+, and Ca2+. In haze-fog environment, both the equivalent salt deposit density (ESDD) and non-soluble deposit density (NSDD) of insulators are higher than that under general environment. The amount of large particles on the AC transmission line is greater than that of the DC transmission line. The influence of DC polarity power source on the distribution of contamination particle size is not significant. After the deposition of haze-fog, the local conductivity of the room temperature vulcanized silicone rubber surface increased, which caused the flashover voltage reduce. Discharge is liable to occur at the triple junction point of droplet, air, and room temperature vulcanized silicone rubber surface. After the deformation or movement of droplets, a new triple junction point would be formed, which would seriously reduce the dielectric strength of room temperature vulcanized silicone rubber.

Detection and localization of partial discharge are very important in condition monitoring of power cables, so it is necessary to build an accurate recognizer to recognize the discharge types. In this paper, firstly, a power cable model based on FDTD simulation is built to get the typical discharge signals as training samples. Secondly, because the extraction of discharge signal features is crucial, fractal characteristics of the training samples are extracted and inputted into the recognizer. To make the results more accurate, multi-SVM recognizer made up of six Support Vector Machines (SVM) is proposed in this paper. The result of the multi-SVM recognizer is determined by the vote of the six SVM. Finally, the BP neural networks and ELM are compared with multi-SVM. The accuracy comparison shows that the multi-SVM recognizer has the best accuracy and stability, and it can recognize the discharge type efficiently.

To elucidate the mechanisms of fruit body development in H. marmoreus, a total of 43609521 high-quality RNA-seq reads were obtained from four developmental stages, including the mycelial knot (H-M), mycelial pigmentation (H-V), primordium (H-P) and fruiting body (H-F) stages. These reads were assembled to obtain 40568 unigenes with an average length of 1074 bp. A total of 26800 (66.06%) unigenes were annotated and analyzed with the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Eukaryotic Orthologous Group (KOG) databases. Differentially expressed genes (DEGs) from the four transcriptomes were analyzed. The KEGG enrichment analysis revealed that the mycelium pigmentation stage was associated with the MAPK, cAMP, and blue light signal transduction pathways. In addition, expression of the two-component system members changed with the transition from H-M to H-V, suggesting that light affected the expression of genes related to fruit body initiation in H. marmoreus. During the transition from H-V to H-P, stress signals associated with MAPK, cAMP and ROS signals might be the most important inducers. Our data suggested that nitrogen starvation might be one of the most important factors in promoting fruit body maturation, and nitrogen metabolism and mTOR signaling pathway were associated with this process. In addition, 30 genes of interest were analyzed by quantitative real-time PCR to verify their expression profiles at the four developmental stages. This study advances our understanding of the molecular mechanism of fruiting body development in H. marmoreus by identifying a wealth of new genes that may play important roles in mushroom morphogenesis.

Equivalent salt deposit density (ESDD) and non-soluble deposit density (NSDD) measurements are a basic requirement of power systems. In order to predict the site pollution severity (SPS) of insulators, a new method based on random forests (RFs) is proposed. Using mutual information (MI) theory and RFs, the weights of factors related to the SPS of insulators are analyzed. The samples of contaminated insulators are extracted from the transmission lines of high voltage alternating current (HVAC) and high voltage direct current transmission (HVDC). The regression models of RFs and support vector machines (SVM) are constructed and compared, which helps to support the lack of information in predicting NSDD in previous works. The results are as follows: according to the mean decrease accuracy (MDA), mean decrease Gini, (MDG), and MI, the types of the insulators (including surface area, surface orientation, and total length) as well as the hydrophobicity are the main factors affecting both ESDD and NSDD. Compared with NSDD, the electrical parameters have a significant effect on ESDD. For the influence factors of ESDD, the weights of the insulator type, hydrophobicity, and meteorological factors are 52.94%, 6.35%, and 21.88%, respectively. For the influence factors of NSDD, the weights of the insulator type, hydrophobicity, and meteorological factors are 55.37%, 11.04%, and 14.26%, respectively. The influence voltage level (vl), voltage type (vt), polarity/phases (pp) exerted on ESDD are 1.5 times, 3 times, and 4.5 times of NSDD, respectively. The influence that distance from the coastline (d), wind velocity (wv), and rainfall (rf) exert on NSDD are 1.5 times, 2 times, and 2.5 times that of ESDD, respectively. Compared with the natural contamination test and the SVM regression model, the RFs regression model can effectively predict the contamination degree of insulators, and the relative error of the predicted ESDD and NSDD is 8.31% and 9.62%, respectively.

Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum.

Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.