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.

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>.

Effects of Exogenous Substances on Growth of Polyporus umbellatus Mycelium and Its Polysaccharide Content / 中国实验方剂学杂志

Objective:To explore the effects of diverse exogenous substances at different concentrations on the growth of<italic> Polyporus umbellatus</italic> mycelium and polysaccharide content and screen out the optimal growth condition for <italic>P. umbellatus</italic> mycelium， so as to provide a reference for its large-scale artificial cultivation. Method:<italic>P. umbellatus</italic> mycelium was cultured in media containing different exogenous substances using the method for fungal culturing in plate. The growth rate of the mycelium was judged by the colony diameter and the polysaccharide content was determined by the phenol-sulfuric acid method. Result:The high-dose cyclic adenosine monophosphate， 6-benzyl aminopurine （6-BA）， gibberellic acid （GA）， 2，4-dichlorophenoxyacetic acid （2，4-D）， vitamin （V） B₁， VB₃， VB₆， VB₉， and VB₁₂ all promoted the growth of <italic>P. umbellatus</italic> mycelium and elevated polysaccharides content. By contrast， indole acetic acid （IAA）， VC， and VB₂ inhibited its growth， with the most obvious inhibition detected in the high-dose VC group. IAA and VB₂ both reduced the polysaccharide content， whereas the high-dose VC significantly increased the polysaccharide content. Cyclic adenosine monophosphate， 6-BA， GA， 2，4-D， VB₁， VB₃， VB₆， VB₉， and VB₁₂ at the concentrations of 2 mmol·L^-1， 6 mg·L^-1， 15 mg·L^-1， 2 mg·L^-1， 4 mg·L^-1， 2 mg·L^-1， 4 mg·L^-1， 6 mg·L^-1， and 10 mg·L^-1， respectively， contributed to the growth of <italic>P. umbellatus</italic> mycelium<italic> </italic>and polysaccharide accumulation. Conclusion:The growth of <italic>P. umbellatus </italic>mycelium and polysaccharide accumulation can be regulated by adding exogenous substances to the culture medium.

New polyketides from chemical mutant strain of sponge-associated fungus emericella variecolor xsa-07-2 / 中国药学杂志

OBJECTIVE: To study the new bioactive secondary metabolites of diethyl sulfate chemical mutant strain of sponge-associated fungus Emericella variecolor XSA-07-2. METHODS: Diethyl sulfate was used to make chemical mutagenesis of strain XSA-07-2, and one mutant strain M8 was chosen for large-scale fermentation to generate new secondary metabolites. The compounds were isolated and purified by chromatography on silica gel and ODS reversed-phase column and semi-preparative HPLC techniques. And their structures were identified by their physicochemical properties and NMR, MS data analysis. RESULTS: Three new polyketides 1-3 were isolated from the extract of the solid fermentation culture of mutant strain M8. Compound 3 showed moderated antioxidant activity with IC50 of (13.58±0.14) μg·mL-1 by DPPH assay. CONCLUSION: Diethyl sulfate chemical mutagenesis can stimulate sponge-associated fungus Emericella variecolor XSA-07-2 mutant strain M8 to produce new antioxidant polyketides.