Cytotoxic sesquiterpene aryl esters from Armillaria gallica 012m / 中草药·英文版

OBJECTIVE@#To study the chemical constituents of the EtOAc extract of Armillaria gallica 012m.@*METHODS@#The chemical constituents of the EtOAc extract of A. gallica 012m were isolated and purified by various column chromatography and their structures were elucidated on the basis of the 1D and 2D NMR spectroscopic and HRESIMS data. Cytotoxicity of all isolates against A549, HCT-116, M231 and W256 human tumor cells was determined by the MTT method.@*RESULTS@#A new sesquiterpene aryl ester, armimelleolide C ( 1), and eight known ones including armillarivin ( 2), melleolide F ( 3), 6'-chloromelleolide F ( 4), melleolide ( 5), melleolide K ( 6), melledonol ( 7), 13-hydroxydihydromelleolide ( 8), and armillane ( 9), were isolated from the EtOAc extract of A. gallica 012m. All isolates showed potential cytotoxic activities against at least one of the human cancer cell lines with IC50 values ranging from (3.17 ± 0.54) to (17.57 ± 0.47) μmol/L. Compound 1 showed significant inhibitory activity against M231 with an IC50 value of (7.54 ± 0.24) μmol/L compared with paclitaxel as the positive control. Compounds 2, 3, and 7, 9 showed obvious inhibitory activity against HCT-116 and were better than that of the positive control.@*CONCLUSION@#The chemical constituents including a new sesquiterpene aryl ester armimelleolide C ( 1) from the EtOAc extract of A. gallica 012m have a variety of structures and potential antitumor activities.

Screening of an Armillaria gallica strain for Gastrodia elata cultivation / 中国中药杂志

This study aims to screen a strain from Armillaria for the cultivation of Gastrodia elata. Specifically, Armillaria strains were isolated from different producing areas of G. elata and identified. Based on the growth characteristics of the strains and the experiment on the cultivation of G. elata, an optimal A. gallica strain was screened out. The specific process is as follows. The fungus-gro-wing materials of G. elata were collected from four producing areas and the Armillaria strains were isolated(G,Y,S,H). The strains were then identified based on morphological observation and phylogeny analysis and the commonly used strains were determined. The sucrase genotypes of the strains were identified according to our previous research findings, and the growth characteristics of the strains, such as growth rate, diameter, dry weight, and polysaccharide content of the rhizomorphs, were measured. According to the biological characteristics and sucrase genotypes, two strains were selected for the cultivation of G. elata. The tuber yield and the content of gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata were measured to select the optimal strain. The results showed that the four strains were all A. gallica. The rhizomorphs of strains G and H of the same sucrase genotype had larger/higher length, growth rate, diameter, branch number, dry weight, and polysaccharide content than those of strains S and Y of the same sucrase genotype. The tuber yield and the total content of gastrodin and p-hydroxybenzyl alcohol in tuber of G. elata cultivated with strain H were 6.528 kg·m~(-2) and 0.566%, respectively, which were 4.58 and 1.30 folds those of G. elata cultivated with strain S. Strains H and S were screened out from four strains of A. gallica based on the growth characteristics and sucrase genotype. According to the tuber yield and content of total gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata, strain H was identified as the optimal one. The findings in this study are expected to lay a basis for cultivating G. elata with high yield and quality of tubers.

Optimization of Agrobacterium-Mediated Genetic Transformation System of Armillaria gallica / 中国实验方剂学杂志

ObjectiveTo optimize the existing genetic transformation system of Armillaria gallica to improve the transformation efficiency and lay a foundation for the follow-up research on Armillaria molecular marker-assisted breeding and gene function. MethodThe genetically transformed plasmid pH101-PAgGPD-GFP-TrpC was constructed，transformed into Escherichia coli，amplified， and cultured，and the plasmid was extracted. The extracted plasmid was transformed into four different agrobacteria LBA4404，EHA105，GV3101，and AGL-1，respectively. The transformed agrobacteria were used for impregnating A. gallica，and the agrobacteria with the highest conversion rate were screened out. Then the agrobacterium-mediated genetic transformation system of A. gallica was optimized from the type and concentration of antibiotics，co-culture time，concentration of bacterial solution， and impregnation method. The phenotype profiles of A. gallica under different conditions were observed using Synbiosis ProtoCol 3. ResultThe optimized genetic transformation conditions of A. gallica were as follows： the Agrobacterium strain of EHA105 at absorbance A600 nm=0.6， the co-culture time of 2 d， the infection mode of negative pressure impregnation for 10 min， the primary screening medium of PDA medium containing 400 mg·L-1 cefotaxime sodium and 10 mg·L-1 hygromycin，and the secondary screening medium of PDA medium containing 12 mg·L-1 hygromycin. ConclusionIn this study，the existing genetic transformation system of A. gallica was optimized，and there was a significant difference in the transformation rate before and after optimization （P<0.05）. After optimization，the transformation efficiency of A. gallica was about 4.33%，which was about eight times higher than that before optimization.

Utilization of used fungus-growing materials of Gastrodia elata / 中国中药杂志

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

A Soybean Straw Cultivation Medium for Armillaria gallica and Optimal Composition / 中国实验方剂学杂志

Objective:To explore the feasibility of replacing wood （or wood chips） with crop residues for culturing <italic>Armillaria gallica</italic> targeting the problems of forest resource destruction and increased cultivation cost caused by the extensive use of wood in <italic>Gastrodia elata</italic> cultivation， so as to reduce the cultivation cost of <italic>G. elata</italic>， promote the effective use of crop residues， and protect forest resources. Method:The growth situation of <italic>A. gallica</italic> in different media was observed， followed by the measurement of its growth rate using streaking method and the determination of total polysaccharide content of <italic>A. gallica</italic> by phenol-concentrated sulfuric acid colorimetric method. In order to further optimize the soybean straw cultivation medium， we carried out a four-factor three-level L₉（3⁴） orthogonal assay on the ratio of main ingredients， sucrose content， inorganic salt content， and water content. Result:The comparison of growing states of <italic>A. gallica</italic> cultured in different media revealed that <italic>A. gallica</italic> in soybean straw medium began to grow since the fourth day of inoculation， and the mycelium grew well， with the growth rate being 0.352 cm·d^-1， which was 1.48 times that in birch wood medium. The total polysaccharide content of <italic>A. gallica</italic> cultured in soybean straw medium was the highest， which was 39.260 mg·g^-1， much higher than 17.028 mg·g^-1 of that cultured in birch wood medium. This demonstrated the obvious advantage of soybean straw medium， whose main ingredients were soybean straw and wheat bran at the ratio of 8：2， with the sucrose and inorganic salt content accounting for 1% and 0.5% of the main ingredients， respectively. When the water content reached 50%， the growth rate of <italic>A. gallica</italic> was maintained at 0.392 cm·d^-1. Conclusion:This study has provided a basis for utilizing soybean straw instead of wood （or wood chips） as cultivation medium for <italic>A. gallica</italic>， thus better reducing the waste of forest resources and protecting the natural environment in the cultivation of <italic>G. elata</italic>.

Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in Armillaria gallica / 中国实验方剂学杂志

Objiective： In the process of microRNA expression analysis by quantitative Real-time polymerase chain reaction（Real-time PCR），the selection of miRNA plays an important role in data standardization. Method:In this paper，13 Armillaria gallica.Candidate miRNAs were selected for bioinformatics analysis of their precursors，and the PMRD was used to predict similar sequences of their precursors，and the RNAfold was used to predict the secondary structure of the candidate miRNAs and their similar sequences. Real-time PCR was used to detect miRNAs expression in two genotypes of Armillaria gallica（genotype A，genotype B） before and after salt stress，and geNorm，NormFinder and BestKeeper were used to analyze the stability of miRNAs expression. Result:Secondary structure prediction and characterization of 9 candidate miRNA precursors showed that the miRNA predicted belonged to the miR family with typical stem-loop structure and the mature miRNAs were at the 5' or 3' end of the miRNA precursors.geNorm analysis showed that genotype A Armillaria gallica could select Novel-4* and Novel-9 as reference gene，genotype B could select Novel-9 and Novel-16 as its reference gene.NormFinder analysis showed that Novel-9 was stable in both genotype A and B Armillaria gallica.BestKeeper analysis showed that Novel-12* was stable in genotype A Armillaria gallica and Novel-2* was stable in genotype B Armillaria gallica. Conclusion:miRNA Novel-9 is the best stable reference gene，which lays a foundation for further research on the regulation mechanism of miRNA in Armillaria gallica.

Application of SmartRoot in Phenotype Profile Analysis of Armillaria gallica / 中国实验方剂学杂志

Objective:To establish a research platform for obtaining accurate phenotypic spectrum data is a technical difficulty that needs to be resolved in the research of traditional Chinese medicine resources. For example，the traditional phenotypic characterization method of Armillaria rhizomorph is mostly in a form of descriptive text，which is subjective and empirical. There is an urgent need for an objective and accurate method to characterize the phenotype of honey fungus rhizomorph. Method:Based on the image processing software Image J and the root identification plug-in SmartRoot combined with the Synbiosis ProtoCol 3 image analyzer，the growth picture of Armillaria spp. was analyzed，and the length，growth rate，branching situation，and angle of nascent rhizomorph of Armillaria gallica were measured to establish a measurement system for the phenotypic analysis of Armillaria rhizomorph. Result:Based on the method developed in this paper，the growth length，growth rate，number of branches，angle of nascent rhizomorph，and other phenotypic changes can be analyzed in a real-time manner without affecting the growth of Armillaria gallica. Armillaria spp. grew fastest at 9-12 days after generation，and the angle between the nascent rhizomorph and the parent rhizomorph was nearly vertical. This method had a certain correlation with the dry weight of traditional Armillaria biomass phenotypic parameters，with a high value in practical application. Conclusion:This study has established an objective，accurate，fast and real-time phenotypic analysis and measurement system for Armillaria rhizomorph，which expands the scope of application of SmartRoot and can be used for phenotypic analysis of traditional Chinese medicine resources under controlled experimental conditions.

Effects of High Temperature Stress on Growth Characteristics of Armillaria / 中国实验方剂学杂志

Objective:To explore the effects of high temperature stress on the growth characteristics of different Armillaria strains，and to provide guidance for screening excellent Armillaria strains with high-temperature resistance. Method:14 strains of Armillaria from different G. elata producing areas were used as experimental materials to observe the growth characteristics and conduct phenotypic classification for the strains. rDNA-IGS sequence analysis was used for molecular identification to further determine the genetic relationship of the tested strains.The strain growth rate, biomass，mycelial length and other indicators under the condition of 23 ℃ （CK） and 30 ℃ high temperature stress were recorded. Result:All the 14 strains of Armillaria had the highest similarity and the closest relationship with Armillaria gallica，but there were significant differences in growth characteristics among different G. elata producing areas. The 14 strains of Armillaria were classified into Ⅳ groups，and the growth status was groupⅠ>group Ⅱ>group Ⅲ>group Ⅳ. After treatment with high temperature stress，the tolerance of each strain to high temperature also showed obvious differences，as shown in the average growth rate of the mycelial was GZ16>SX1>GZ1. The rank of relative mycelial length was GZ16>SX1>GZ3 and the relative biomass was GZ16>SX4>GZ1>HB1>AH2. Conclusion:Under high temperature stress，GZ16 was best in growth rate，relative length of mycelial，relative biomass and growth state，followed by SX1 and GZ1 strains. The results indicate that strains GZ16，SX1 and GZ1 have the strong resistance to high temperature and excellent growth characteristics at normal temperature，so these three strains are suitable to be produced in main G. elata producing areas in China.

Effect of Gastrodia elata Planting on Soil Microbial Community Structure / 中国实验方剂学杂志

Objective:To analyze the changes of soil microbial community structure before and after planting Gastrodia elata in different producing areas，and to investigate the response of soil microorganisms to the planting of G. elata. Method:ITS and 16S rDNA high-throughput sequencing technologies were used to detect fungal and bacterial community compositions in the soil，including the soil without planting G. elata（CK1，CK2），the soil around G. elata（GE1，GE2）before harvesting, and the soil around the rhizomorph of Armillaria（AGE1，AGE2） in Dafang, Guizhou and Jinzhai, Anhui respectively. Result:Principal component analysis (PCA) showed that the soil microorganisms changed significantly after G. elata planting as compared with the control soil. The sequencing results showed that the planting of G. elata increased the OTUs number of fungi and bacteria. As compared with the control soil，the diversity and abundance of fungal and bacterial communities showed an increase trend after the cultivation of G. elata in soil of Dafang, Guizhou， such changes of fungal communities were not significant, but the abundance of soil bacteria communities increased in Jinzhai, Anhui as compared with the control soil. The abundance of genera Ilyonectria and Nitrospira increased，while genera Russula decreased significantly both in the soil of Guizhou and Anhui. Furthermore，the abundance of Fusarium and Mortierella increased significantly in the soil of Dafang, Guizhou. Conclusion:The soil microorganisms were out of balance after planting of G. elata, and the abundance of pathogenic microorganisms such as Ilyonectria and Fusarium increased，which may be related to the plant diseases and insect pests of G. elata.

Bioinformatics analysis and expression of chitinase genes in Armillaria gallica / 中国中药杂志

Armillaria gallica is a facultative parasitic fungus which is the only nutrient source of Gastrodia elata during its cultivation.Chitinase,as a glycosidic hydrolytic enzyme,plays an important role in the growth,development,stress tolerance and symbiotic signal transduction of A. gallica. There were 22 chitinase genes in A. gallica. Bioinformatics analysis of amino acid sequence of these chitinase genes revealed that 12 chitinase genes contained glycosidase 18 family( GH18) domain. Chitinase amino acid sequences of A. gallica,A. ostoyae,G. elata,Saccharomyces cerevisiae and Trichoderma harzianum were analyzed byclustering trees,so as to further predict the gene function of chitinase in A. gallica. Induction of A. gallica branching with strigolactone analogue GR24,high-throughput sequencing technology based on the induction of branch group( MHJ1),uninduced branch group( MHJ2) and blank control group( MHJ3) is used to detect the expression quantity,the transcription level data of 22 chitinase genes were obtained and the heat map was generated for expression pattern analysis. It was found that 8 genes may be involved in physiological processes such as A. gallica branching,cell wall degradation and remodeling. In this paper,the function of chitinase gene in A. gallica was just preliminarily analyzed and predicted.

Molecular identification of Armillaria gallica by PCR-RFLP analysis / 中国中药杂志

Armillaria gallica is a symbiotic fungus in the cultivation process of Gastrodia elata and Polyporus.The rhizomorph of A. gallica invades the stalk of the G. elata or the Sclerotium of the Polyporus,and is digested and utilized by the latter,becoming their important source of nutrition. Different nature of A. gallica affects the growth of G. elata and Polyporus. The authors collected A. gallica from 13 commercially available regions and screened two A. gallica,A and B,at the genetic and metabolic levels,in order to distinguish between the two A. gallica market. We have established convenient and effective DNA molecular identification method.By comparing the sequence differences between the A. gallica type A and type B invertase genes,PCR-RFLP primers were designed based on differential fragment. Primer ZTM.F/ZTM.R can amplified A. gallica type A and B,producing a band of about 304 bp in length. The restriction endonuclease EcoR V could recognize the difference sequence of A and B types of A. gallica. The type B was digested to form two fragments,thereby specifically identifying the A. gallica as type B. The established methods of PCR-RFLP is an accurate identification method for A. gallica. Therefore,in the cultivation process of G. elata and Polyporus,suitable strains can be selected according to different needs of variety,growth stage and ecological environment,and the yield and quality can be improved according to local conditions.

Transcriptome analysis on symbiotic molecular mechanism of Armillaria mellea and Gastrodia elata / 中草药

Objective In this study, Armillaria mellea and Zhaotong Gastrodia elata vegetative stems were used as the experimental material to reveal the symbiosis molecular mechanism of A. mellea infecting G. elata through comparative transcriptome sequencing analysis. Methods Trizor reason (invitrogen) was used to extract RNA from vegetative propagation stem samples, and the sequencing library was established and sequenced. Results The results showed that 38 838 sequences were annotated when G. elata symbiosis with A. mellea. Under false discovery rate (FDR) 1 screening conditions, there were significant differences in expression levels among the 23 333 genes, in which 1 595 genes up-regulated and 21 738 down-regulated expression. Based on the transcriptome analysis, unigenes associated with the extracellular enzyme gene cellulase, xylanase, laccase, and polygalacturonase genes were 21, 6, 39, and 6 genes, respectively, in which the number of corresponding differentially expressed genes were 9, 3, 23, and 4, respectively. The expression of all these unigenes were down-regulated except for one cellulase genes. The number of unigenes related to anti-oxidant enzymes superoxide dismutase, peroxidase, and catalase were 13, 7, and 17, respectively. The differentially expressed genes respectively were 6, 7, and 7, all of which were down-regulated. In addition, these genes were selectively confirmed by real-time PCR. Conclusion The life activites of A. mellea which infect and symbiosis with G. elata declined, meanwhile G. elata did not produce biological stress for A. mellea. This study provides a lot of valuable genetic resources for further studying the symbiosis molecular mechanism of A. mellea and G. elata.

Analysis of monosaccharide composition of twelve polysaccharides by pre-column derivatization procedure UPLC-MS/MS / 中国中药杂志

This study is to establish a pre-column derivatization procedure with 1-phenyl-3-methyl-5-pyrazolone (PMP) UPLC-MS/MS method for the determination of the monosaccharide composition of 12 polysaccharides. At the same time, the monosaccharide components of polysaccharides in Armillaria gallica were analyzed. The separation was performed on a ACQUITY ZORBAX RRHD Eclipse Plus C₁₈ column(2.1 mm×100 mm, 1.8 μm),using 95% acetonitrile (A) and ammonium acetate-5% acetonitrile-water (B) as mobile phase with gradient elution. The target components were detected in multiple-reaction monitoring (MRM) mode by mass spectrometry with electrospray ionization (ESI) source operated in ionization mode. The results showed that based on the monosaccharides detection method established by UPLC-MS/MS, the linearity of the 12 monosaccharides components were linear in their linear range (R²>0.990), and the recovery rate were 92.30%-105.6%. 11 monosaccharides such as fructose, mannose, and glucose were detected in A. gallica samples. The method established in this experiment is robust, highly reproducible and accurate, and is suitable for the determination of monosaccharide components such as A. gallica.

Correlative analysis advance of chemical constituents of Polyporus umbellatus and Armillaria mellea / 中国中药杂志

Medicinal Polyporus umbellatus is the dry sclerotia of P. umbellatus, with the effect of diuresis; Armillaria mellea is a parasitic fungus which can infect plants up to 300 genera, with sedative, anticonvulsant and some other biological activities. As the medicinal value of P. umbellatus and A. mellea is increasingly wide concerned, the market quantity demanded of them is gradually increased and the demand outstrips the supply. The symbiotic A. mellea and P. umbellatus are both the medicinal and edible fungi with diverse activities, including hypoglycemic action, improve immunity and antitumor and so on. The growth of the sclerotia forming from the mycelium of P. umbellatus is related to the infection of the symbiotic A. mellea and their secondary products. In this study, by comparing the chemical constituents of the mycelium and sclerotia of P. umbellatus and A. mellea, we found that they all produced steroids and nitrogen-containing heterocycles. The sclerotia of P. umbellatus and A. mellea also produced triterpenes secondary metabolites. In addition, the mycelium and infected sclerotia of P. umbellatus mainly produced different steroids, and the sclerotia produced some other special secondary metabolites, such as long-chain fatty acids, ceramides, phenol and so on. By analyzing above all kinds of differences, speculated that these may be caused by the infection of the symbiotic A. mellea which mainly produced sesquiterpenes, diterpenes and other secondary metabolites. The contents and types of compounds of P. umbellatus and A. mellea are closely related to their symbiosis and reproduction, therefore, many symbiosis mechanisms should be found by utilizing more molecular biology technology to elucidate this complex symbiotic infection and provide scientific basis for improving the yield and quality of P. umbellatus and A. mellea.

Research progress in chemical constituents and biological activities of Armillaria mellea / 中草药

Armillaria mellea is a kind of medicinal and edible fungus belonging to Tricholomataceae family. It possesses comprehensive biological activities, such as hypnotic, sedative, improving cardiocerebral blood circulation, hypoglycemic, antioxidant, and immune regulation. Besides, it can inhibit tumor. A. mellea contains characteristic chemical constituents, such as, protoilludane sesquiterpenoid aromatic esters, diterpenoids, triterpenoids, polysaccharides, adenosine, and various types of secondary metabolites. This paper summarizes the chemical composition and biological activity of A. mellea to provide the reference for developing and utilizing it.

Chemical constituents from water extract of Armillaria luteo-virens / 中国药科大学学报

@#A two-dimensional preparative liquid chromatographic method was developed for purification of constituents from water extract of Armillaria luteo-virens using reversed phase preparative liquid chromatography(RPLC)coupled with hydrophilic interaction liquid chromatography(HILIC). Seven compounds were isolated from fruit bodies of Armillaria luteo-virens, and their structures were identified on the basis of physicochemical and spectroscopic analyses. These compounds were identified as pyroglutamic acid(1), uridine(2), 2′-deoxyuridine(3), uracil(4), guanosine(5), inosine(6), and adenosine(7). All the compounds were purified and identified from fruit bodies of Armillaria luteo-virens for the first time.

Investigation of symbiotic Armillaria species with Chinese traditional medicinal fungus Polyporus umbellatus / 中国药学杂志

OBJECTIVE: To identify symbiotic Armillaria species with Polyporus umbellatus. METHODS: Armillaria was cultured on PDA paltes. To determine whether the strains were pure culture, the internal intergenic spacer (IGS) region were sequenced. Furthermore, the NCBI BLAST program was used to search similar sequences in the GenBanksequence database for the IGS sequence of the species on homology, and a phylogenetic tree was constructed based on neighbor-joining method. RESULTS: All isolates were similar in colony morphology and grew well in PDA medium with well-developed rhizomorphs. The isolated 22 strains were pure culture of the fungus. Phylogenetic analysis based on IGS sequence indicated that the symbiotic Armillaria species with Polyporus umbellatus belonged to A. cepistipes, A. gallica, A. ostoyae, and A. mellea. CONCLUSION: In the present study, phylogenetic analysis based on molecular method was carried out for the symbiotic Armillaria species with P. umbellatus for the first time. The results suggest that Armillaria species have no specificity with P. umbellatus, which is different from the previous reports. Further research will be done on the growth of P. umbellatus affected by different kinds of Armillaria species.

Purification and characterization of a ribonuclease from the wild edible mushroom Armillaria luteo-virens.

A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70°C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U)>poly(C)>poly (G)>poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.

Anti-aging mechanism of polysaccharide from rhizomorph of Armillaria mellea in Caenorhabditis elegans / 中草药

Objective: To explore the anti-aging mechanism of polysaccharide from rhizomorph of Armillaria mellea (AMP) in Caenorhabditis elegans. Methods: With C. elegans as living model, the life span and the average number of offsprings were determined under normal culture conditions; The survival rate under oxidative stress and the expression of heat shock protein-16.2 (HSP-16.2) and superoxide dismutase-3 (SOD-3) were determined. Results: Under the normal culture conditions, the life span of C. elegans was significantly extended by AMP-1 and AMP-2 without the damage of the reproductive capacity; The expression of HSP-16.2 and SOD-3 was increased in C. elegans under the oxidative stress. Conclusion: The possible mechanism of AMP for anti-aging of C. elegans may be caused by increasing of the capacity of stress resistance.

The Fruiting Body Formation of Armillaria mellea on Oak Sawdust Medium Covered with Ground Raw Carrots

To produce an artificial fruiting body of Armillaria mellea on the oak sawdust medium, seven strains of A. mellea were used. The top surface of oak sawdust medium covered with ground raw carrot was inoculated with each of 7 strains and cultured for 30 days at 25degrees C in the dark condition until the mycelia of A. mellea completely colonized the medium from top to bottom. Then, the mycelia which were fully covered on the top surface of the medium were scratched slightly with a spatula and filled with tap water for 3 hours. To induce the primordial formation, the 7 strains of A. mellea were transferred to the growth chamber under the illumination (350 lux) of 12 hours and relative humidity of 85 +/- 5% in a day and then cultured at 16 +/- 1degrees C. Only A. mellea IUM 949 could form primordia on the sawdust medium, but the other strains did not make primordia at the same condition. The primordia of A. mellea IUM 949 were formed 10 days after complete colonization of the medium and the fruiting bodies were produced 7 days after a primordial formation. The experimental results suggested that IUM 949 strain might be a good candidate for mass production of fruiting bodies of A. mellea.