Circular RNA is a class of covalently closed RNAs, which plays important roles in the regulation of gene expression, such as competitive endogenous RNAs, protein scaffolds, and translation templates. The processes for identifying circular RNAs in fungi are reviewed for the purposes of providing theoretical support for unravelling the regulatory role of circular RNA in fungal developments and metabolisms.
Based on morphological, anatomical, chemical and molecular data, a new species, Gypsoplaca albimarginata, collected from Bayinbulak Nature Reserve, is described. The new species is characterized by having white edged thallus with rhizoid and reddish-brown apothecium embedded in thallus. The species is described and illustrated, and its distribution and the difference between the species and other affined species are briefly discussed.
In recent years, stipe rot disease caused heavy losses in Morchella sextelata cultivation. In order to clarify the pathogen, 12 samples of diseased Morchella sextelata fruiting bodies from Sichuan, Henan, Gansu, and Guizhou were collected. Thirty-seven fungal strains and three bacterial strains were isolated from these samples. According to the Koch’s rule, GJB-3 was confirmed as the pathogenic strain. Based on ITS, RPB2 and EF1α sequences analyses, colony morphology and microscopic structures, GJB-3 was identified as Fusarium nematophilum. Prevention and control of stipe rot disease on Morchella will be further studied.
Arbuscular mycorrhizal fungal (AMF) diversity in Cinnamomum camphora seedlings bred with root production method (RPM) and conventional technology (hereinafter as ‘conventional seedlings’) in Longfu Experimental Base of Hunan Academy of Forestry Sciences was studied. AMF samples were collected from roots and rhizosphere soil of the two kinds of seedlings, and identification was based on morphology and high-throughput sequencing. The results showed the roots of Cinnamomum camphora seedlings bred with the two methods manifested arbuscular mycorrhizal characteristics, and no ectomycorrhizal structure was found. A total of 25 738 valid sequences belonging to 1 phylum, 2 classes, 3 orders, 5 families and 5 genera of AMF was obtained. According to the proportion of valid sequence ranged from high to low the five genera were Acaulospora, Glomus, Diversispora, Rhizophagus and Paraglomus. The dominant genera in RPM seedlings and conventional seedlings were Acaulospora and Glomus, but the proportion of Glomus in RPM seedlings was higher (28.04% and 57.36% respectively in root and rhizosphere soil) and the proportion of Acaulospora in conventional seedlings was higher (99.58% and 42.2% respectively in root and rhizosphere soil). For both seedlings, the Ace index and Chao index in rhizosphere soil were much higher than those in roots. The richness index of AMF in rhizosphere soil of RPM seedlings was higher than that of conventional seedlings, however, there was no significant difference in AMF richness index in the root between the two kinds of seedlings (P<0.05). Redundancy analysis showed that soil pH and available phosphorus were the main factors affecting AMF community structure, and different physical and chemical factors had different effects on AMF community.
Illumina MiSeq high-throughput sequencing technology was used to examine the effects of three different cropping treatments (rotation of paddy rice and Brassica oleracea var. italica, rotation of pumpkin and B. oleracea var. italica, and continuous cropping of B. oleracea var. italica) on the fungal community structure and diversity in the rhizosphere of B. oleracea var. italica cultivated in the coastal beach area in Taizhou Bay, Zhejiang Province, China. A total of 11 phyla, 27 classes, 52 orders, 94 families, and 155 genera of soil fungi was isolated. Ascomycota, Olpidiomycota, Basidiomycota, Zygomycota, and Chytridiomycota were the dominant phyla with high relative abundance. At the phylum level, rotation cropping notably improved the relative abundance of Ascomycota and Chytridiomycota but decreased that of Olpidiomycota and Zygomycota in B. oleracea var. italica rhizosphere soil. At the genus level, rotation cropping decreased the relative abundance of Olpidium and Fusarium, whereas increased that of Pleospora in B. oleracea var. italica rhizosphere soil. Paddy rice-upland crop rotation very significantly heightened diversity and richness indices of rhizosphere fungi, while upland crop rotation and B. oleracea var. italica continuous cropping took less effect. Compared with long-term continuous cropping of B. oleracea var. italica, paddy rice-upland crop rotation and upland crop rotation could improve the fungal community structure in B. oleracea var. italica rhizosphere. In particular, paddy rice-upland crop rotation had better effects on improving the abundance and diversity of beneficial fungi and decreasing those of harmful fungi in B. oleracea var. italica rhizosphere, enhancing the ecological environment of the rhizosphere. These findings provide practical evidence of achieving high yield and excellent quality of B. oleracea var. italica through rotation cropping.
Based on resequencing five strains of Lentinula edodes and mapping the whole genome sequence of monokaryon strain L808-1, the insertion/deletion (InDel) markers on 10 chromosomes of L. edodes genome were analyzed. Loci with more than 15 InDel bases were selected, and 449 pairs of InDel primers were synthesized. PCR and electrophoresis results revealed that 237 pairs among them showed clear bands, and 107 pairs (PIC≥ 3) were screened out as core InDel markers, which were later applied to analyze the genetic diversity of 44 L. edodes strains. Cluster analysis grouped the cultivars and wild strains into individual branches respectively, indicating the obvious population stratification among them. Population structure analysis categorized the germplasm resources of L. edodes into four subgroups. Principal component analysis showed that the position and distance between strains were consistent with the results of cluster analysis and population structure analysis. The InDel markers developed and applied in this paper provide a basis for the construction of L. edodes core germplasm and effective breeding of germplasms.
A CRISPR/Cas9 gene editing system was successfully constructed in nematode-trapping fungus Arthrobotrys oligospora YMF 1.03170 by optimizing the sgRNA expression driving system tRNAGly. The CRISPR/Cas9 system combined with homologous recombination led to the precise replacement of the two target amino acid coding genes at the same time. Combined with metabolic profile and feeding experiment of precursor compounds, new active sites Arg17, Arg18, His33 and His34 in 6-methylsalicylate synthase 283 were found. The fine gene editing system CRISPR/Cas9 established in Arthrobotrys oligospora provided an effective method for rapidly constructing the genetic transformation system of Arthrobotrys oligospora and studying the gene function of this fungus.
Morels, members of the genus Morchella of the phylum Ascomycota, are among the most highly prized edible mushrooms worldwide. Genomic sequencing of Morchella sextelata strain SCLS that is successfully cultivated in Sichuan Province of China was performed by using PacBio single-molecule long-read sequencing technology. A high-quality genome assembly containing 53.57Mb sequences with a repeat content of 17.03% was generated. The SCLS strain genome assembly comprises 42 contigs with a N50 attaining the length of 1.82Mb. Among them, 13 contigs have telomeric repeats on both ends, representing full-length chromosomes. A total of 13 182 protein-coding genes was identified by strand-specific RNA-seq sequencing and transcript assembling together with multiple gene prediction strategies, of which 267 genes encoding carbohydrate-active enzymes and 11 secondary metabolite biosynthetic gene clusters were identified. Comparison with the genome of the M. sextelata strain NZTD180501373 cultivated in Inner Mongolia revealed that chromosomal recombination events might have occurred during the evolution of M. sextelata. There are 33 055 SNP and 48 726 InDel site differences between the two strains and each strain has over 6Mb specific sequences. Comparing with M. importuna, some of orthogroups related to transposase have expanded in SCLS strain and the SCLS strain has additional 5 unique transposase gene families with more than 100 members in each family. DNA N6-methyladenine (6mA) modification in the genome of M. sextelata SCLS was identified. The results showed that 0.42% of adenine in the strain SCLS genome was modified by 6mA and its content was significantly higher than that in 6 reported dikarya fungi. The 6mA sites are enriched on the LTR retrotransposon, indicating that 6mA methylation in M. sextelata may regulate the activity of LTR retrotransposon. This is the first report that DNA 6mA methylation is associated with transposons in fungi.
In order to study the signal mechanism of 9,10-cyclomethylheptadecanoic acid (9,10-CMA) - induced triterpenoid acid (TA) synthesis in Ganoderma lingzhi, the mediating role of NO signal was analyzed. The results showed that under the induction of 9,10-CMA, NO could effectively trigger the activation of phenylalanine ammonia lyase (PAL) and cytochrome CYP450, and the synthesis of triterpenoid acids in G. lingzhi mycelia. Nitric oxide plays an inducer as a upstream molecule of CYP450 and PAL activating in G. lingzhi cell, but it is not the only signal molecule. The dynamic expression of six key enzymes in triterpenoid acid synthesis induced by 9,10-CMA in G. lingzhi was analyzed. The results showed that sqs, CYP5150L8 and hmgr genes related to triterpenoid acid synthesis in G. lingzhi were significantly up-regulated under 9,10-CMA induction, indicating that these three corresponding synthetases may play critical roles in the induction process.
A UDP-glucose-4-epimerase (UGE) gene, which plays a key role in the synthesis of polysaccharides, was obtained through multi-omics analysis of the mutated strain Hericium erinaceus 321, and was heterologously expressed in E. coli BL21 (DE3). The inductive expression conditions were optimized. The high purity target protein was obtained after purification by nickel column affinity chromatography. The enzymatic properties of the protein were studied, and the optimal activity temperature and pH were determined. The biological properties and kinetic parameters of the protein were defined. The results provided theoretical reference for development and utilization of the gene.
Enhancement of laccase production from the Trametes sp. strain LS-10C by adding the rare earth element of yttrium was investigated. The yttrium element rendered a notable promoting effect on the laccase production, and the laccase production level reached the maximum activity of 70.25U/mL when the concentration of Y3+ in the medium was 12mmol/L, being 5.3 times higher than that obtained in the medium without addition of Y3+. Proteomics analysis showed that Y3+mainly promoted the upregulation of membrane fraction-related protein expression, and the number of the upregulated proteins accounted for 70.5% of the total number of the upregulated proteins in the cellular fraction enrichment. In addition, obvious enrichments of the upregulated proteins in both metabolic processes of carbohydrate and oxalate were observed. This study clarified that the rare earth element has a promoting effect on the laccase production of Trametes, and preliminarily analyzed the proteome changes of Trametes under the action of Y3+, which laid a substantial foundation for studying the molecular mechanism of rare earth element effect on laccase production of Trametes sp.
The study of Agrobacterium tumefaciens EHA105 mediated transformation of Pestalotiopsis microspora KFRD-2 was carried out by using recombinant vector pCAMBgfp containing hygromycin resistance gene and GFP gene. Based on hygromycin and GFP fluorescence resistance, the transformants were screened. Through the analysis of colony morphology, mycelium growth rate, sporulation, fluorescence stability and pathogenicity verification, more than 100 positive transformants were screened out. The average transformation efficiency was up to 200 transformants per 106conidia. Most transformants were no significant differences in morphological characteristics and pathogenicity as compared with the wild-type strains. Fourteen positive mutants with significant differences in mycelial morphological characteristics, sporulation or pathogenicity were obtained, which could be used to explore the virulence genes and the pathogenic mechanism of P. microspora.
Trolox equivalent antioxidant capacity (TEAC) of ethanol extracts from 38 different batches and growth age of fruiting bodies of Sanghuangporus baumii were analyzed. There were differences in TEAC of different batches, but no significant difference was observed in that of different growth age. HPLC fingerprint of S. baumii alcohol extract was established, and 27 common peaks (P1-P27) were obtained. Based on orthogonal partial least square (OPLS) regression, the number of Latent Variables was 3; the cumulative model predictive parameters (Q2) were 0.803; the variable cumulative explanatory indexes (R2X) and (R2Y) were 0.574 and 0.954, respectively; the square of correlation coefficient (R2) was 0.954. The results showed that good prediction effects were obtained, and the correlation between peak and activity could be accurately reflected. By using the variable importance for the projection (VIP) index of model variables, 14 common peaks were obtained, which contributed significantly to the antioxidant activity. The model of spectrum effect relationship of Sanghuangporus baumii can assist the separation and purification of active compounds, evaluation of the medicinal activity value, and establishment of quality standards of fruiting bodies.
Polysaccharide is one of the main pharmacological active components of Ganoderma lingzhi. The preventive effect of polysaccharides from G. lingzhi fruit-body (GLFPS) on acute alcoholic liver injury in mice was analyzed by detecting serum indexes, and the mechanism of metabonomics was explored. The results showed that GLFPS significantly inhibited the levels of ALT, AST, TG, TC and ADH in serum of mice induced by alcohol. With the help of metabonomics analysis, 85 different metabolites were obtained between the model group and the control group. Among them, adenosine triphosphate, L-aspartic acid, and L-lysine play important roles in the interaction network, indicating that alcohol can cause changes in the metabolism of adenosine and amino acids in the liver of mice. There were 58 different metabolites between the GLFPS treated group and the model group, mainly including lipids and organic oxygen. KEGG enrichment analysis of different metabolites showed that they were involved in choline metabolism, glycerophospholipid metabolism, and ABC transporters. GLFPS can effectively relieve hurt of these three metabolic pathways significantly changed by alcohol. In conclusion, GLFPS effectively prevent alcoholic liver injury by regulating the metabolism of choline, glycerophospholipids, and some ABC transporters in mice.
Artificially cultivated Isaria cicadae fruiting bodies (ACICF) (with dosages of 0.85, 1.70, 3.40g/kg) were continuously orally administered for 10 days to Wistar rats on the 7th to 16th day of pregnancy. The effects on pregnancy body weight, stillbirth rate, absorbed fetal rate, number of live fetuses, average number of fetuses per litter, fetal weight, fetal length, bregma width, uterine including fetal weight, fetal appearance, internal organ development, and fetal bone development were investigated to study the possibility of teratogenicity of pregnant animals contacting ACICF. Meanwhile, different doses of ACICF (0.4, 0.25, 0.125g/kg) were orally administered to normal mice and STZ-induced hyperglycemia model mice, and fasting blood glucose and glucose tolerance were measured to evaluate hypoglycemic effect of ACICF. The results showed that each ACICF-treated group had no significant difference as compared with the negative control group in the weight, stillbirth rate, absorbed fetal rate, fetal mouse weight, fetal mouse body length and bregma width, fetal mouse appearance, and development of internal organ and bone (P>0.05). Compared with the model control group (0g/kg BW), both the medium and low dose ACICF-treated groups could reduce the blood glucose level of STZ-induced hyperglycemia model mice in 0.5h after administration of glucose (P<0.05), and the low dose treatment could significantly reduce the area under the 0-2h blood glucose curve of hyperglycemia model mice. ACICF had no adverse effects on the weight of the mice and the fasting blood glucose of normal mice. It is suggested that ACICF has no obvious toxicity and teratogenicity to maternal rats and embryo and it has a hypoglycemic effect on hyperglycemic mice.
Stropharia rugosoannulata was selected as the test material, and the effects of different concentrations of selenium (0, 6, 15, 20mg/L) on the growth characteristics of mycelia under cadmium stress (2mg/L) were analyzed. Results showed that compared with the untreated control, the growth rate and biomass of S. rugosoannulata under cadmium treatment were inhibited, but the osmotic adjustment substances, hydrogen peroxide and malondialdehyde content increased significantly. Under combined treatment of selenium and cadmium, the growth rate and biomass of S. rugosoannulata mycelia were improved, and osmotic adjustment substances were significantly increased, promoting the osmotic adjustment ability of S. rugosoannulata under cadmium stress. Selenium induced up-regulation of antioxidant enzyme activities, improved the ability of scavenging active oxygen, reduced the content of malondialdehyde and hydrogen peroxide in the mycelia, relieved the degree of peroxidation of the cell membrane, and enhanced the tolerance of S. rugosoannulata to cadmium stress. This research focuses on the effects of selenium on the growth, physiological characteristics and anti-oxidative stress of S. rugosoannulata under cadmium stress, and theoretically lays a basis for further applying selenium in cadmium-resistant cultivation of edible fungi.
Comparisons of morphology, amino acid profile and protein quality between strain with ball-shaped fruiting bodies and normal strain of Lentinula edodes were conducted. Based on the latest version of international amino acid reference patterns, amino acid score (AAS), IOM (Institute of Medicine) pattern score, chemical score (CS), ratio coefficient of amino acid (RC), score of ratio coefficient of amino acid (SRC) and essential amino acid index (EAAI) were used to evaluate their nutrition value. Morphologically, the stipe and gill of the strain with ball-shaped fruiting bodies were not differentiated, and the nutrition of the strain also changed. In terms of crude protein content, the ball-shaped strain was 1.37 times higher than the normal strain (32.32% and 23.60%, respectively). In terms of the average total amino acid content, the ball-shaped strain was 1.28 times higher than the normal strain (209.58mg/g and 163.10mg/g, respectively). The ball-shaped strain could be hopefully used as the breeding material for screening new cultivated strains.
The chemical constituents and anti-tumour activity in vitro of cultivated Pholiota adiposa were studied by using UPLC-QTOF-MS method. HepG-2, A549, HeLa and MCF-7 cells were cultured in vitro and treated with different concentrations of ethanol extracts of Pholiota adiposa (EPA), and the cytotoxicity was assayed by CCK-8 method. Flow cytometry was used for HepG-2 cells, apoptosis analysis via annexin VFITC/PI double staining. A total of 38 components is consistant with that recorded in the chemical composition database. The content of palmitic acid and 9E,12E-octadecadienoic acid was the highest, and sterol compounds and polysaccharide compounds accounted for a relatively high proportion. EPA showed the strongest cytotoxicity to HepG-2 cells, and the apoptosis rate was up to (23.71±1.59)% under the concentration of 50μg/mL. Pholiota adiposa has a variety of functional chemical constituents and potential anti-tumor application value. It is preliminally concluded that the anti-tumor activity is realized by the functional constituents through inducing apoptosis.
Protective effects of different doses (100, 200 and 400mg/kg) of water extract (WE), water extract after extraction by ethanol (WEE) and ethanol extract (EE) of Taiwanofungus camphoratu on alcohol-induced acute liver injury in ICR mice, as well as Nrf2/HO-1 antioxidant signaling pathway were investigated. Compared with the model group, 400mg/kg dosage of both WE and WEE could inhibit the increase of serum ALT and AST levels significantly, and 200mg/kg of WE and WEE could obviously decrease the serum ALT and AST content, respectively. Different doses of WE, WEE and EE could significantly reduce the content of hepatic MDA; 200 and 400mg/kg of WE and different doses of WEE could markedly increase hepatic SOD and CAT activities. H&E staining observation indicated that WE, WEE and EE could mitigate alcoholic liver injury to a certain degree, but a relative lower effect was observed in EE-treated groups. The immunohistochemical staining results indicated that different dose of WE, WEE and EE could enhance the nuclear translocation of Nrf2 and promote the expression of HO-1 to improve the hepatic antioxidant capacity, thus presenting a significant protective effect on alcohol-induced acute liver injury. It was suggested that Taiwanofungus camphoratus exhibited drunkenness-alleviating and liver-protecting effects via regulating the Nrf2 antioxidant signaling pathway.
Triterpene is one of the most important active ingredients in Ganoderma lingzhi. Previous studies found that oleic acid could promote the biosynthesis of triterpenes in liquid submerged fermentation of Ganoderma lingzhi. In this study, the process of liquid submerged fermentation for synthesis of triterpenes by using oleic acid as promoter was optimized and verified on the scale of a 3L fermenter. The effects of the additive method, time for addition and concentration of oleic acid on triterpene synthesis were investigated by single factor experiment. The optimal process conditions were obtained and verified by response surface experiment. The highest triterpene content reaching 42.69mg/g was obtained by adding 1.21% moist heat sterilized oleic acid at 32h of fermentation, and by adding 1.35% filtered sterilized oleic acid at the 7h of fermentation, the highest triterpene content was 43.38mg/g, being 2.04 times and 2.08 times higher than triterpene content of the control, respectively. The triterpenes content was 32.18mg/g and 32.48mg/g respectively by adding moist heat sterilized and filterer sterilized oleic acid at 1 000mL scale of shake culture, being 1.96 times and 1.95 times higher as compared with the control. The triterpene content in 3L fermenter was 28.66mg/g and 25.13mg/g, 1.62 times and 1.42 times higher than that of the control. The technological conditions of liquid submerged fermentation of triterpenes promoted by oleic acid were successfully optimized and verified in a 3L fermenter, corresponding to industrial production. This study provide an important reference for large-scale fermentation of Ganoderma lingzhi for production of triterpenes.
Trametes lactinea is a macrofungus with rich bioactive substances. In this study, on the basis of single factor experiment, Box‐Behnken design and response surface methodology the optimization of extraction process of polysaccharide of the fungus was carried out. The crude polysaccharides were separated and purified by DEAE-Cellulose-52 anion exchange column and Sephadex G-200 chromatography column, and uniform polysaccharide components TLMPS-0, TLMPS-1 and TLMPS-3 were obtained. The structures of the three polysaccharides were analyzed by chemical composition analysis, UV-vis, FTIR and Congo red experiments, and the free radical scavenging ability and reducing power of the polysaccharides were also tested. The results showed that the optimal extraction conditions were as follows: extraction temperature of 99°C, solid material/liquid ratio of 1:30 (g/mL), extraction duration of 5h, successive extraction of 2 times. Under this condition, the extraction yield of polysaccharide was 4.01%. The content of uronic acid of TLMPS-0, TLMPS-1 and TLMPS-3 was 12.91%±0.44%, 8.24%±0.22% and 7.50%±0.66%, respectively, and the content of sulfuric acid group was 22.24%±1.88%, 14.55%±0.56% and 18.68%±0.69%, respectively. It is shown that TLMPS-0 is an α-gyranoid polysaccharide or β-gyranoid polysaccharide. The TLMPS-1 is a β-gyranoid polysaccharide, and three polysaccharides do not have a tricotic structure. All the three polysaccharides have certain ability of scavenging DPPH free radical, ABTS free radical and hydroxyl radical and reducing iron ion, among which TLMPS-0 has the strongest antioxidant activity. The results provided theoretical basis for functional research of polysaccharides of T. lactinea.
Hot reflux extraction process was optimized for Ganoderma lingzhi triterpenoids based on ultra-high performance liquid-triple quadrupole mass spectrometry (UPLC-MS/MS) quantitative detection method. The established UPLC-MS/MS method was used to precisely detect the content of 19 triterpenoids of G. lingzhi fruiting bodies of seven cultivated varieties from different sources, and fruiting body of sd-2 G. lingzhi with high triterpenoids content was selected as the extractive material. Based on triterpenoid content and extract yield, the triterpenoid extraction process was optimized through single factor and response surface experiments. The best optimization conditions obtained were as follows: 75% of ethanol concentration, 2.5h of extraction time, 14:1 of liquid-to-material ratio, and two times of repeated extraction. A pilot experiment was conducted for obtained optimal extraction technology, and the yield of the ethanol extract of G. lingzhi fruiting body was 6.10%, and the content of triterpenoid was 11.8591mg/g. The results provide theoretical reference for the industrialized large-scale extraction of G. lingzhi triterpenoids and technical support of further development and utilization of G. lingzhi high value-added products.
Different methods were used to degrade Ganoderma lingzhi β-glucan, and the degradation rate, molecular weight variation and polymerization degree distribution were used as indexes to compare the degradation effects. Results showed that the degradation rates of the β-glucans treated by microwave, enzymatic and chemical methods were about 94%, 40% and 20%, respectively. By use of microwave method, the molecular weight of degradation products significantly reduced, and the polymerization degree of oligosaccharides was widely distributed. The distribution of oligosaccharide polymerization degree in β-glucan degradation products obtained through enzymatic treatment was narrow, and the oligosaccharide products under acid and alkali treatments were less. Among four tested methods, microwave exhibited the highest efficiency, and the operation was simple and controllable, being suitable for the preparation of Ganoderma lingzhi β-glucooligosaccharide.
Agaricus sinodeliciosus ‘Shenmei 1’ was bred and domesticated from wild strains. Demonstrative cultivation for years shows that ‘Shenmei 1’ has the characteristics of medium temperature growth (growing well at 23°C, and fruiting at 16-18°C), high yield (with biological efficiency of 50%), and short cultivation period (53d). The fruiting bodies are round-shaped, with compact quality, rich flavor and high genetic stability. It is suitable for bagged cultivation and industrialized facility cultivation.
Pleurotus pulmomarius ‘Hangxiu 2’ was bred from ‘Taixiu 57’ strain by multi spore selfing. The suitable temperature for mycelial growth and fruiting body growth is 26-28°C and 22-28°C, respectively. The cap is fan-shaped and dark gray, with an average size of 4.38cm×3.65cm; The stipe is cylindrical, white, with an average diameter of 1.06cm. The protein content of fresh fruiting body was 2.94%, and the total amino acid content was 2.54%. The demonstration cultivation showed that the average biological efficiency of ‘Hangxiu 2’ is 71.2%. After low temperature stimulation, one crop could be harvested in 5-6 days. This variety has the characteristics of good marketability and uniform fruiting. It is suitable for high temperature and off-season cultivation.
