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  • CRISPR/Cas9-Assisted Hybridization Strategy for Generating Homozygous Mutants at Targeted Locus in Ganoderma lucidum
    TIAN Jialin, DONG Beibei, TANG Chuanhong, ZHOU Shuai, FENG Na, FENG Jie, ZHANG Jingsong, TAN Yi
    2025, 32(3): 1-12. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.001
    Abstract ( ) PDF ( ) Knowledge map Save
    Using the auxotrophic screening marker gene ura3 as the target, CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing was carried out on dikaryotic Ganoderma lucidum strain G0119 (cultivar ‘Hunong Lingzhi No.1’) and its mating-compatible monokaryotic strains L1 and L2. The editing efficiencies between the monokaryotic and dikaryotic strains were compared. The edited monokaryotic strains of L1 and L2 were crossed to generate dikaryotic strains with homozygous mutations in ura3. The results showed that seven edited strains of G0119 were obtained. Crossing of these edited G0119 strains with edited strains of L1 and L2 showed that all seven edited G0119 strains were heterozygous: six of them were edited in their L1-type nuclei, and the remaining one was edited in its L2-type nuclei. There were 10 and 7 monokaryotic edited strains of L1 and L2, respectively, and the editing efficiency of L1 nuclei was 100%. Crossings of monokaryotic edited strains L1-2×L2-1, both having a C-base deletion in ura3, and monokaryotic edited strains L1-5×L2-3, both containing an A-base insertion in ura3, generated two dikaryotic homozygous ura3 disrupted strains. This is a showcase of obtaining dikaryotic G. lucidum mutants with homozygous mutations in targeted genes through CRISPR/Cas9 ribonucleoprotein-mediated gene editing and single hybridization. This strategy also provided a new approach for phenotypic analysis of dikaryotic G. lucidum strains by enabling precise genetic manipulation of targeted genes.
  • Transcriptome Analysis of Mechanism of Gamma-Aminobutyric Acid Biosynthesis in Flammulina filiformis During Fruiting Body Development
    LI Wenyun, LU Xuewei, SHAO Youran, WANG Ruijuan, SHANG Xiaodong, FENG Zhan, LI Hewen, WU Yingying
    2025, 32(3): 13-27. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.002
    Abstract ( ) PDF ( ) Knowledge map Save
    Gamma-aminobutyric acid (GABA), ubiquitously exists in edible fungi, is a non-protein natural amino acid with important physiological functions. The content of GABA is high in Flammulina filiformis. To understand the biosynthetic mechanism of GABA in F. filiformis, samples of F. filiformis fruiting bodies at 15 d, 21 d and 25 d after scratching were analyzed for transcriptome data, with 15-21 d defined as elongation stage and 21-25 d defined as mature stage. Samples of different collection time were also measured for contents of GABA and its precursors so as to identify genes related to GABA biosynthesis. RT-qPCR and addition of key substances in the biosynthesis pathway were used to verify the identified genes. The results showed that GABA and its synthetic precursors, glutamate and ornithine, initially increased and then decreased as the fruiting bodies elongate and mature, whereas the content of arginine kept increasing. Compared with the samples of 15 d, there were 869 and 641 differentially expressed genes (DEGs) at the elongation and mature stages, respectively. GO functional enrichment analysis showed that the DEGs were mainly enriched in secondary units such as putrescine catabolism, polyamine catabolism, ornithine biosynthesis, and glutamate metabolism. KEGG metabolic pathway analysis showed that the DEGs were mainly enriched in metabolism of various amino acids and biosynthesis of steroids and folate. By RT-qPCR validation and functional prediction analysis, scaffold1.g1430, scaffold1.g390, scaffold6.g197, scaffold9.g519, and scaffold11.g88 were potential important genes related to GABA biosynthesis in F. filiformis, and they were also involved in the synthesis of various precursors in the GABA shunt and polyamine degradation pathway. The addition of 0.5 g·L-1 L-glutamic acid and 0.2 g·L-1 CaCl2 to liquid medium increased GABA content in mycelia of F. filiformis, suggesting that the GABA shunt may be involved in GABA biosynthesis in F. filiformis. These findings provided a reference for elucidating the GABA metabolic pathway in F. filiformis.
  • Analysis of Phenylalanine Ammonia-Lyase Gene Expression and Its Effect on Pileus Color in Pleurotus eryngii
    NIE Menghan, CHEN Lili, LUO Liyuan, ZHANG Tuo, XIE Baogui
    2025, 32(3): 28-37. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.003
    Abstract ( ) PDF ( ) Knowledge map Save
    Pleurotus eryngii strain Pe0100 was analyzed for phenylalanine ammonia-lyase (PAL) gene sequences, and expression pattern of PAL genes across five developmental stages, mycelium, primordium, young mushroom, elongation, and maturation. In particular, the expression of PAL genes in pileus and stipe was compared at the young mushroom, elongation and maturation stages. Using Pe0100 as the control, one of the PAL genes in P. eryngii, PePAL1, was overexpressed in Pe0100 by transformation of the overexpression plasmid Pe-PAL1. The PePAL1 overexpression transformants were then observed for phenotypic characteristics and analyzed for correlation between PAL activity and PAL gene expression in mature pileus. The results showed that there were two PAL genes, PePAL1 and PePAL2, in the genome of Pe0100. Compared with stipe, pileus showed higher expression of PePAL1 at the young mushroom, elongation and maturation stages. On the other hand, PePAL2 showed a higher expression level than PePAL1 in mycelium, and remained a stable expression level during the rest of the developmental stages. Four PePAL1 overexpression transformants, OE#3, OE#10, OE#16 and OE#18, were obtained. All of them produced dark brown striped pigmentation on the surface of their pilei, and there was a positive correlation between PePAL1 expression and PAL enzyme activity in mature pileus. These results provided a reference for breeding of newP. eryngii cultivars.
  • Transcriptome Analysis of Gene Expression in Morchella sextelata Mycelium under High-Temperature Stress
    HU Xinyu, ZHAI Mengdan, CHEN Zewei, LI Wangqi, HUANG Kaiwen, ZHANG Yuchen, WANG Zhen
    2025, 32(3): 38-50. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.004
    Abstract ( ) PDF ( ) Knowledge map Save
    Using 20 ℃ as the control, the effects of high-temperature (33 ℃) stress on gene expression in mycelia of Morchella sextelata were studied. Comparative transcriptome analysis was used to identify differentially expressed genes (DEGs) between the two experimental conditions. Five randomly selected DEGs were validated by real-time fluorescence quantitative PCR (qPCR), and the correlation between trasnscriptiome data and qPCR results was analyzed by Pearson correlation analysis. The identified DEGs were analyzed for gene ontology (GO) and KEGG metabolic pathway. Based on the screened metabolic pathways, gene set enrichment analysis (GSEA) was carried out to identify genes involved in high-temperature response of mycelia. The results showed that there were 2 112 DEGs between the high-temperature group and the control group. Among them, 799 genes were up-regulated, and 1 313 genes were down-regulated. There was a highly significant positive correlation (R2=0.88) between the qPCR results and the transcriptome sequencing data of the five randomly selected DEGs. The GO functional enrichment analysis showed that the DEGs were primarily enriched in DNA replication, lipid metabolism, membrane components, DNA binding, and drug binding. The KEGG enrichment analysis showed that the DEGs were enriched in several crucial metabolic pathways, such as mismatch repair, ABC transporter, starch and sucrose metabolism, pyruvate metabolism, amino acid biosynthesis, two-component system, RNA degradation, and carbon metabolism.Ten key DEGs were identified to be associated with high-temperature stress, and they were genes encoding DNA helicase, molecular chaperone protein, pyruvate kinase, malolactic enzyme, ATP-binding protein, multidrug resistance efflux pump protein, phosphodiesterase, histidine kinase, transketolase, and fructose-6-phosphate ketolase. These findings provided a reference for elucidating the molecular mechanism underlying response of M. sextelata to high-temperature stress.
  • Changes of Nuclei in Morchella sextelata at Different Developmental Stages
    WANG Xiaomin, ZHANG Qinyu, FAN Yang, ZHANG Bangxi
    2025, 32(3): 51-58. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.005
    Abstract ( ) PDF ( ) Knowledge map Save
    Morchella sextelata was observed for nuclear morphology of ascus, ascospore, primary hypha and secondary hypha by fluorescence microscopy. The results showed that initially there were 10-24 nuclei per ascus, which then moved to the tip and gathered. After cell division, eight oval-shaped ascospores were formed and arranged vertically in a column per ascus. After ascospores were ejected from asci, their volume doubled after cultured for 6 h, and the number of nuclei increased from 9-15 per ascospore to about 21-27 per ascospore. As the cultivation continued, ascospore tip bulged and developed 1-2 germ tubes, which then further elongated and developed septa. There were both single mating type and double mating type in primary hyphae, whereas secondary hyphae were exclusively double mating type. Under microscopic observation, there were differences in nuclear morphology between primary hyphae and secondary hyphae. Primary hyphae typically held 25-52 nuclei per cell, and the nuclei were predominantly granular and small. On the other hand, secondary hyphae typically held 3-10 nuclei per cell, and the nuclei were mostly clumpy and large. Regardless of nuclear shape, both granular and clumpy nuclei were able to transport genetic material between cells through septum.
  • Effect of Precooling on Postharvest Preservation of Volvariella volvacea
    LI Xiaohui, LI Yuanyuan, WANG Wenhan, ZHANG Jingsong, YANG Yan, CHEN Wanchao, JIA Wei
    2025, 32(3): 59-72. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.006
    Abstract ( ) PDF ( ) Knowledge map Save
    Fresh fruiting bodies of Volvariella volvacea V06 were harvested and randomly divided into the following groups: precooling group [cooled by cooling fan to (15±1) ℃ and then stored under (15±1) ℃ and (80±5)% relative humidity], no precooling group [directly stored under(15±1) ℃ and (80±5)% relative humidity after harvest], and room temperature group [directly stored under (23±1) ℃ and (65±5)% relative humidity after harvest]. Fruiting bodies of different treatment groups were then evaluated for sensory characteristics, determined for hardness and browning index, and measured for malondialdehyde (MDA) content and ATP content, polyphenol oxidase (PPO) activity, mitochondrial membrane potential, and tricarboxylic acid (TCA) cycle-related enzyme activities. The results showed that storage under (15±1) ℃ and (80±5)% relative humidity effectively delayed the spoilage of V. volvacea, and precooling further delayed the browning and dehydration of V. volvacea, inhibited the increase of MDA content, and reduced the activity of PPO. The precooling treatment slowed down the decrease of mitochondrial membrane potential, maintained the TCA cycle rate at a relatively stable level, delayed the decrease of activities of mitochondrial complexes Ⅰ, Ⅲ, and Ⅳ, kept ATP at a high level, and prolonged the shelf life of V. volvacea from 1 d to 4 d. These results provided a reference for postharvest preservation of V. volvacea.
  • Biological Characteristics and Domestication of Sanguinoderma ovisporum
    NIU Kaiyang, HAN Lin, LI Zhengcong, FENG Wenzao, LUO Zonglong, TANG Songming
    2025, 32(3): 73-80. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.007
    Abstract ( ) PDF ( ) Knowledge map Save
    Sanguinoderma spp. are edible and medicinal macrofungi rich in various bioactive compounds with antitumor and antioxidant effects. However, the biological characteristics and domestication of Sanguinoderma spp. haven’t been well studied yet. Wild fruiting bodies of S. ovisporum were collected from Dehong, Yunnan Province, and then a strain named FDCU XZ-3 was isolated by tissue isolation. Through single-factor and orthogonal experiments, the effects of different carbon sources, nitrogen sources, inorganic salts, and temperature on mycelial growth rate and mycelium vigor of FDCU XZ-3 were determined. The results showed that the optimal culture conditions for mycelial growth of FDCU XZ-3, i.e., temperature, carbon source, nitrogen source, and inorganic salt, were 28℃, fructose, soy peptone, and sodium chloride, respectively. Fruiting bodies of FDCU XZ-3 were developed on casing soil, and the pileus size, pileus thickness and dry weight per mushroom were (10.20±1.94) cm × (7.36±2.43) cm, (0.4±0.1) cm, and (12.60±4.79) g, respectively. These results provided a reference for cultivation and utilization of S. ovisporum.
  • Antitumor Effect of Pholiota adiposa Water Extract on H22 Tumor-Bearing Mice
    WANG Xiaoyan, LI Gang, KONG Fanli, HE Zhijie, TAN Zhongyu
    2025, 32(3): 81-90. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.008
    Abstract ( ) PDF ( ) Knowledge map Save
    Water extract of artificially cultivated Pholiota adiposa (WPA) was prepared and analyzed for nutritional components. An H22 tumor-bearing mouse model (MC) was established, along with a blank control group (NC, saline by gavage), a positive control group (PC, 25 mg·kg-1 cyclophosphamide), low-dose WPA group (L-WPA, 100 mg·kg-1), medium-dose WPA group (M-WPA, 200 mg·kg-1) and high-dose WPA group (H-WPA, 300 mg·kg-1). After 15 consecutive days of treatment, mice in different groups were measured for tumor suppression rate, organ indices, and contents of IFN-γ, IL-2, IL-6, TNF-α, aspartate aminotransferase (AST), blood urea nitrogen (BUN), and vascular endothelial growth factor (VEGF) in mouse serum by ELISA. Tumor tissues were observed by hematoxylin-eosin (HE) staining, and determined for tumor cell apoptosis by TUNEL assay. The expression of proteins associated with tumor apoptosis, i.e., BAX, Bcl-2, caspase-3, cleaved caspase-3 and VEGF, was determined by immunohistochemistry and Western blot. The results showed that compared with MC, the thymus index and spleen index of M-WPA and H-WPA were significantly increased, the tumor mass of WPA treated mice were extremely significantly decreased, the contents of IL-2, IFN-γ and TNF-α in all WPA groups were significantly increased, and the contents of IL-6, VEGF, AST and BUN in all WPA groups were significantly decreased. Regardless of dose, the tumor apoptosis rates of all WPA groups significantly increased compared with MC. WPA also resulted in significant increases in the expression of BAX, caspase-3, cleaved caspase-3, and significant decreases in the expression of Bcl-2 and VEGF. These results provided a reference for the development and utilization of P. adiposa.
  • Antioxidant Activity of Aqueous Extract of Ganoderma lucidum Mycelia Prepared under a Shaking-Static Cultivation Cycle
    WANG Xing, FENG Jie, JIANG Xingyi, LIU Yanfang, HAN Wei, ZHANG Jingsong
    2025, 32(3): 91-101. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.009
    Abstract ( ) PDF ( ) Knowledge map Save
    Using a shaking-static cultivation cycle of 2.8 d shaking -7.3 d static -0.2 d shaking -0.3 d static, mycelia of Ganoderma lucidum were cultivated in 250 mL shake flasks, 3 L, 5 L, and 50 L bioreactors, respectively. At the same time, continues shaking cultivation at these scales were set as controls. Then mycelia harvested from different cultivation scales were measured for biomass and total triterpene content, and aqueous extracts of mycelia harvested from different cultivation scales were measured for contents of total triterpenes, total phenols, and total sugar. The aqueous extracts were also determined for their activities on scavenging ABTS, DPPH, superoxide anion, and hydroxyl radicals. The results showed that mycelia of shaking-static cultivation groups contained higher amount of total triterpenes compared with the control groups. Under the shaking-static cultivation mode, cultivation in bioreactors achieved higher biomass than cultivation in 250 mL shake flasks, while cultivation at 250 mL scale resulted in the highest total triterpene content among all scales. For aqueous extracts of mycelia obtained under the shaking-static cultivation mode, the aqueous extract of3 L bioreactor cultivated mycelia contained the highest contents of total triterpenes and total sugar, whereas the aqueous extract of 250 mL shake flask cultivated mycelia and that of 3 L bioreactor cultivated mycelia contained high levels of total phenols. In terms of antioxidant activity in vitro, the aqueous extract of 3 L bioreactor cultivated mycelia showed the strongest ABTS radical scavenging activity [IC50 (12.31 ± 0.23) mg·mL-1],while the aqueous extract of 250 mL shake flask cultivated mycelia exhibited the highest activity on scavenging DPPH radicals [IC50 (4.53 ± 0.24) mg·mL-1]. For superoxide anion radicals, the aqueous extracts of mycelia from 3 L and 50 L bioreactors showed relatively strong scavenging activity, with IC50 of(12.29 ± 0.61) mg·mL-1 and (12.66 ± 0.68) mg·mL-1, respectively. For hydroxyl radicals, the aqueous extracts of mycelia from 250 mL shake flasks, 5 L, and 50 L bioreactors showed strong scavenging activity, with IC50 of (12.52 ± 0.73) mg·mL-1, (11.87 ± 0.46) mg·mL-1, and (11.57 ± 0.33) mg·mL-1, respectively. These findings provided a reference for industrialized production and applications of G. lucidum mycelia by liquid fermentation.
  • Effects of Clitocybe squamulosa Polysaccharide on Physicochemical and Structural Properties of Cornstarch
    HOU Shuting, ZHANG Defang, YU Dongmei, WANG Wuxia, XU Yaping, MENG Junlong, CHANG Mingchang, GENG Xueran
    2025, 32(3): 102-112. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.010
    Abstract ( ) PDF ( ) Knowledge map Save
    In order to investigate the effects of Clitocybe squamulosa fruiting body polysaccharide (CSFP) on physicochemical and structural properties of corn starch, CSFP was added to corn starch at 0, 2%, 4%, 6% and 8% based on dry substrate, respectively. These CSFP-cornstarch complexes were then measured for gelatinization properties, rheology properties and texture properties, observed for microstructure by scanning electron microscopy, and analyzed for infrared spectrum. The results showed that CSFP delayed gelatinization of cornstarch, i.e., as the CSFP addition concentration increased, the peak viscosity, minimum viscosity, final viscosity, breakdown viscosity and setback viscosity of cornstarch gelatinization decreased, and the gelatinization temperature increased. The static rheological results showed that both cornstarch paste and the complexes were shear-diluted pseudoplastic fluids, and as the CSFP concentration increased, the fluid index (n) and consistency coefficient (K) increased and decreased, respectively. The dynamic rheological results showed that both cornstarch paste and the CSFP-cornstarch complexes showed weak gel-like behavior. The microstructure of cornstarch turned loose and the internal gaps became larger as a result of the addition of CSFP. After the addition of CSFP, there was no new groups formed in the complexes as shown by infrared spectrum scanning, and the short-range order of the cornstarch structure in the complexes increased. CSFP reduced the hardness, elasticity, cohesiveness, chewiness and stickiness of cornstarch paste, providing a reference for development of easy-to-swallow foods.
  • Determination of Ergosterol in Ganoderma lucidum Spore Oil by HPLC
    WANG Jinyan, TANG Qingjiu, ZHOU Shuai, ZHANG Jingsong, YAN Peilan, ZHOU Jing
    2025, 32(3): 113-119. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.011
    Abstract ( ) PDF ( ) Knowledge map Save
    Through optimization of HPLC conditions, a highly efficient HPLC method for determination of ergosterol in Ganoderma lucidum spore oil was established and then used to determine ergosterol inG. lucidum spore oil samples stored under -20 ℃ or room temperature with or without the presence of vitamin E. The methodological validation results showed that there was a good linear relationship in the concentration range of 2.5-100 μg·L-1 for ergosterol, and the R2 was 0.999 1. The precision, accuracy, stability of the HPLC method was outstanding with a recovery rate of 95.21%-102.07%. This simple, accurate, and stable HPLC method enabled rapid and precise quantification of ergosterol in G. lucidum spore oil, and thus provided a basis for quality assessment of G. lucidum spore oil. The results showed that compared with ergosterol contents of G. lucidum spore oil samples stored at -20 ℃, the ergosterol contents of spoil oil samples from the same batch but stored under room temperature were decreased by 32.1%-47.2% when stored for the same duration. When stored under room temperature, the ergosterol contents of spore oil samples without the addition of vitamin E were 24.1%-28.7% lower than the ergosterol contents of spore oil samples added with vitamin E. These results suggested that frozen storage and vitamin E addition effectively maintained ergosterol content in G. lucidum spore oil, and at the same time provided a reference for establishing a new method for quality evaluation ofG. lucidum spore oil products.
  • Genetic Diversity Analysis and Evaluation of Wild Pleurotus tuoliensis Germplasm Resources
    SONG Shuang, LIU Yu, WU Yueyuan, GAO Qi, WU Lingzhi, ZHANG Xinmei, WANG Shouxian
    2025, 32(3): 120-130. https://doi.org/10.16488/j.cnki.1005一9873.2025.03.012
    Abstract ( ) PDF ( ) Knowledge map Save
    Fifty two wild Pleurotus tuoliensis strains from Tacheng area of Xinjiang province were analyzed for genetic diversity using ISSR markers. They were also observed and measured for agronomic traits (five qualitative and ten quantitative traits), and then evaluated based on the quantitative traits. For genetic diversity analysis, the genomic DNA of the 52 strains were extracted from their mycelia, amplified for ISSR markers, measured for number of alleles, number of polymorphic loci, percentage of polymorphic loci, and number of effective alleles, and calculated for Shannon diversity index and Nei’s gene diversity index. Then a dendrogram of the ISSR sequences of the 52 strains of P. tuoliensis was constructed using the unweighted pair group method with arithmetic mean (UPGMA) algorithm. For the quantitative traits, Q-cluster analysis and principal component analysis were carried out by SPSS16. The results showed that a total of 64 polymorphic loci were amplified by 10 ISSR primers, and the average Nei’s gene diversity index was 0.25. The wildP. tuoliensis strains were categorized into five clusters by UPGMA cluster analysis, and the genetic similarity coefficients were between 0.63 and 0.93. Shannon diversity index for the five qualitative traits ranged from 1.01 to 1.26, and that for the 10 quantitative traits ranged from 1.82 to 2.05, with the largest diversity index of 2.05 for stipe diameter. When the squared Euclidean distance of the Q-cluster analysis of the quantitative traits was 15, the 52 strains were categorized into three groups, including strain JZB2106018 in group 1, strain JZB2106020 in group 2, and the remaining 50 strains in group 3. There was a rich genetic diversity within the 52 strains. Based on the eigenvalues, eigenvectors of each principal component and the standardized quantitative trait data, comprehensive scores were calculated for each strain, and three strains, JZB2106048, JZB2106045 and JZB2106039, showed a high comprehensive score. The single fruiting body weights of the three strains were also relatively large. They can be used as breeding materials or high-quality germplasm resources for P. tuoliensis.
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