Effect of BBM gene on callus growth and ginsenoside content in Panax quinquefolius / 中国中药杂志

Baby Boom(BBM) gene is a key regulatory factor in embryonic development and regeneration, cell proliferation, callus growth, and differentiation promotion. Since the genetic transformation system of Panax quinquefolius is unstable with low efficiency and long period, this study attempted to transfer BBM gene of Zea mays to P. quinquefolius callus by gene gunship to investigate its effect on the callus growth and ginsenoside content, laying a foundation for establishing efficient genetic transformation system of P. quinquefolius. Four transgenic callus of P. quinquefolius with different transformation events were obtained by screening for glufosinate ammonium resistance and molecular identification by PCR. The growth state and growth rate of wild-type and transgenic callus were compared in the same growth period. The content of ginsenoside in transgenic callus was determined by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS). The results showed that transgenic callus growth rate was significantly higher than that of wild-type callus. In addition, the content of ginsenoside Rb_1, Rg_1, Ro, and Re was significantly higher than that in wild-type callus. The paper preliminarily proved the function of BBM gene in promoting growth rate and increasing ginsenoside content, which provided a scientific basis to establish a stable and efficient genetic transformation system for Panax plants in the future.

Application of strigolactone analogs in storage of Gastrodia elata / 中国中药杂志

This study explored the preservation effect of strigolactone analogs on Gastrodia elata tubers and screened out the suitable preservation measures of G. elata to provide a safer and more effective method for its storage and preservation. Fresh G. elata tubers were treated with 7FGR24, 2,4-D isooctyl ester, and maleic hydrazide, respectively. The growth of flower buds, the activities of CAT, and MDA, and the content of gastrodin and p-hydroxybenzyl alcohol were measured to compare the effects of different compounds on the storage and preservation of G. elata. The effects of different storage temperatures on the preservation of 7FGR24 were compared and analyzed. The gibberellin signal transduction receptor gene GeGID1 was cloned, and the effect of 7FGR24 on the expression level of GeGID1 was analyzed by quantitative polymerase chain reaction(qPCR). The toxicity of the G. elata preservative 7FGR24 was analyzed by intragastric administration in mice to evaluate its safety. The results showed that compared with 2,4-D isooctyl ester and maleic hydrazide, 7FGR24 treatment had a significant inhibitory effect on the growth of G. elata flower buds, and the CAT enzyme activity of G. elata was the highest, indicating that its preservation effect was stronger. Different storage temperatures had different effects on the preservation of G. elata, and the preservation effect was the strongest at 5 ℃. The open reading frame(ORF) of GeGID1 gene was 936 bp in length, and its expression level was significantly down-regulated after 7FGR24 treatment, indicating that 7FGR24 may inhibit the growth of flower buds by inhibiting the gibberellin signal of G. elata, thereby exerting a fresh-keeping effect. Feeding preservative 7FGR24 had no significant effect on the behavior and physiology of mice, indicating that it had no obvious toxicity. This study explored the application of the strigolactone analog 7FGR24 in the storage and preservation of G. elata and preliminarily established a method for the storage and preservation of G. elata, laying a foundation for the molecular mechanism of 7FGR24 in the storage and preservation of G. elata.

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.

Comparative and phylogeny analysis of Asarum caudigerum and A. cardiophyllum complete chloroplast genomes / 药学学报

The complete chloroplast genome of medicinal plant Asarum caudigerum Hance and its close relative A. cardiophyllum Franchet were sequenced using Illumina Hiseq technology, and assembled, annotated, and characterized by bioinformatic methods in this study. Then phylogenetic analysis of the complete chloroplast genomes of A. caudigerum, A. cardiophyllum, and twelve published species was conducted. The results indicated that the chloroplast genomes ranged from 186 215-186 985 bp in length, with a large single copy (LSC, 89 445-90 169 bp) and two inverted repeats (IRa/IRb, 48 387-48 408 bp). The overall GC content was 37.4%-37.5%. A total of 144 chloroplast genes were annotated, including 98 protein coding genes, 38 tRNA genes and 8 rRNA genes. In addition, complex genomic rearrangements were detected in the chloroplast genome of Asarum. Meanwhile, visual evaluation of the discrete type of the sequence indicated that the variation level of non-coding region was higher than that of coding region. Phylogenetic analyses suggested that A. caudigerum and A. cardiophyllum were clustered into a single clade and A. cardiophyllum, A. sieboldii var. seoulense, A. misandrum and A. maculatum were clustered into another single branch. These two clade were sister species. This study provides a scientific basis for the identification, phylogenetic relationship, molecular breeding of Asarum species.

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.

Bioinformatics analysis and function prediction of ginseng TCP transcription factor family / 中国中药杂志

The longevity mechanism of ginseng(Panax ginseng) is related to its strong meristematic ability. In this paper, this study used bioinformatic methods to identify the members of the ginseng TCP gene family in the whole genome and analyzed their sequence characteristics. Then, quantitative real-time fluorescent PCR was performed to analyze the TCP genes containing elements rela-ted to meristem expression in the taproots, fibrous roots, stems, and leaves. According to the data, this study further explored the expression specificity of TCP genes in ginseng tissues, which facilitated the dissection of the longevity mechanism of ginseng. The ginseng TCP members were identified and analyzed using PlantTFDB, ExPASy, MEME, PLANTCARE, TBtools, MEGA and DNAMAN. The results demonstrated that there were 60 TCP gene family members in ginseng, and they could be divided into two classes: Class Ⅰ and Class Ⅱ, in which the Class Ⅱ possessed two subclasses: CYC-TCP and CIN-TCP. The deduced TCP proteins in ginseng had the length of 128-793 aa, the isoelectric point of 4.49-9.84 and the relative molecular mass of 14.2-89.3 kDa. They all contained the basic helix-loop-helix(bHLH) domain. There are a variety of stress response-related cis-acting elements in the promoter regions of ginseng TCP genes, and PgTCP20-PgTCP24 contained the elements associated with meristematic expression. The transcription levels of PgTCP20-PgTCP24 were high in fibrous roots and leaves, but low in stems, indicating the tissue-specific expression of ginseng TCP genes. The Class Ⅰ TCP members which contained PgTCP20-PgTCP23, may be important regulators for the growth and development of ginseng roots.

Genetic diversity of protopine-6-hydroxylase in three medicinal Papaver plants / 中国中药杂志

Sanguinarine is the main active component of the Papaver plants, and protopine-6-hydroxylase(P6 H), involved in the sanguinarine biosynthetic pathway, can oxidize protopine to 6-hydroxyprotopine. The investigation on the diversity of P6 H genes in the medicinal Papaver plants contributes to the acquirement of P6 H with high activity to increase the biosynthesis of sanguinarine. Five P6 H genes in P. somniferum, P. orientale, and P. rhoeas were discovered based on the re-sequencing data of the Papaver species, followed by bioinformatics analysis. With the elongation factor 1α(EF-1α), which exhibits stable expression in the root and stem, as the internal reference gene, the transcription levels of P6H genes in roots and stems of the Papaver plants were detected by real-time fluorescent quantitative PCR. As indicated by the re-sequencing results, there were two genotypes of P6H in P. somniferum and P. orientale, respectively, and only one in P. rhoeas. The bioinformatics analysis showed that the P6 H proteins of the three Papaver plants contained the conserved domain cl12078, which is the characteristic of p450 supergene family, and transmembrane regions. The existence of signal peptide remained verification. Real-time fluorescent quantitative PCR results revealed that the transcription level of P6 H in roots of P. somniferum was about 1.44 times of that in stems(α=0.05). The present study confirmed genetic diversity of P6 H in the three medicinal Papaver plants, which lays a basis for the research on the biosynthesis pathway and mechanism of sanguinarine in Papaver species.

Differences between male and female leaves of Schisandra sphenanthera: based on RNA-Seq / 中国中药杂志

Schisandra sphenanthera is dioecious and only the fruits of female plants can be used as medicine and food. It is of great significance for the cultivation and production of S. sphenanthera to explore the differences between male and female plants at the non-flowering stage and develop the identification markers at non-flowering or seedling stage. In this study, the transcriptome of male and female leaves of S. sphenanthera at the non-flowering stage was sequenced by Illumina high-throughput sequencing technology and analyzed based on bioinformatics. A total of 236 682 transcripts were assembled by Trinity software and 171 588 were chosen as unigenes. Finally, 1 525 differentially expressed genes(DEGs) were identified, with 458 up-regulated and 1 067 down-regulated in female lea-ves. The down-regulated genes mainly involve photosynthesis, photosynthesis-antenna protein, carbon fixation in photosynthetic or-ganisms, and other pathways. Real-time quantitative PCR(qPCR) identified two genes between male and female leaves and one of them was a HVA22-like gene related to floral organ development and abscisic acid(ABA). Enzyme linked immunosorbent assay(ELISA) was applied to determine the content of ABA, auxin, gibberellin, and zeatin riboside(ZR) in leaves of S. sphenanthera. The results showed that the content of ABA and ZR in male leaves was significantly higher than that in female leaves. The involvement of down-regulated genes in female leaves in the photosynthesis pathway and the significant differences in the content of endogenous hormones between male and female leaves lay a scientific basis for analyzing the factors affecting sex differentiation of S. sphenanthera.

Effects of Dimethyl Sulfoxide and Ethanol on Adventitious Root Growth of Panax quinquefolium and Ginsenosides / 中国实验方剂学杂志

Objective:To screen out the suitable nonpolar molecular cosolvent and concentration with adventitious root phenotype and ginsenoside content in the controlled experiment as the evaluation indexes， so as to lay a solid foundation for exploring the causes for good shape and high quality of <italic>Panax quinquefolium</italic>. Method:After being treated with different concentrations of dimethyl sulfoxide （DMSO） and ethanol， the adventitious roots were scanned using a panoramic scanner， and the resulting images were used for measuring the branch number and average diameter by WinRHIZO Pro 2016， Synbiosis ProtoCol 3 colony counter， Image J， and SmartRoot. The contents of ginsenosides Rg₁， Rb₁， and Re were determined by ultra-performance liquid chromatography tandem mass spectrometry （UPLC-MS/MS）. Result:Compared with the blank control， the 0.1% DMSO and 75% ethanol made the adventitious root phenotype and ginsenoside contents significantly changed. Specifically， the branch number and average diameter were significantly reduced. The ginsenoside Rg₁ in the adventitious roots decreased after 0.1% DMSO treatment， whereas the ginsenosides Rg₁ and Re increased after 75% ethanol treatment. The adventitious root phenotype and ginsenoside contents in the 0.1% DMSO treatment group were not significantly different from those in the control group. Conclusion:The 0.01% DMSO does not affect the adventitious root growth of <italic>P. quinquefolium </italic>and is insoluble in water， enabling it to be considered as a suitable nonpolar molecular cosolvent for future research on the genetic causes for the good shape and high quality of <italic>P. quinquefolium</italic>.

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.

Quantitative Classification of Murraya paniculata Leaf Traits and Endogenous Hormone Contents Analysis / 中国实验方剂学杂志

Objective:To carry out germplasm resource evaluation and new variety breeding of <italic>Murraya paniculata</italic> and improve the germplasm quality， so as to ensure the demand， yield and quality of medicinal materials. Method:Following resource investigation and collection， 17 traits of 107 <italic>M. paniculata</italic> germplasm samples， like plant type， basal diameter， leaf shape， leaf length， and leaf width were determined and then subjected to principal component analysis and factor analysis for screening the principal component factors. Nine primary traits were selected as variables for further cluster analysis using Ward's method and Euclidean distance. According to the characteristics of medicinal parts， the core germplasms were screened out. Then the contents of auxin， zeatin， zeatin nucleoside， isopentenyl adenine， isopentenyl adenine riboside， dihydrozeatin， and dihydrozeatinriboside in the leaves were measured by ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）， followed by their correlation analysis with agronomic trait. Result:The variation coefficients of petiole length， branching number， and basal diameter were large. The nine main factors could be classified into four categories， with a contribution rate of 72.822%. The cluster analysis with Ward's method and Euclidean distance showed that 107 germplasm samples were clustered into six clusters and 61 core germplasms were identified. Such traits as leaf length， leaf width， petiole length， leaf surface， and petiole color were found to play an important role in the classification of <italic>M. paniculata</italic> germplasms. The content of zeatin nucleoside exhibited significant positive correlations with leaf length （<italic>P</italic><0.01）， petiole length （<italic>P</italic><0.01）， and leaf width （<italic>P</italic><0.05）. Conclusion:These results have laid the foundation for further selection and breeding of <italic>M. paniculata</italic> new varieties.

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.

Research Progress of Endophytic Bacterial Resources and Their Secondary Metabolites / 中国实验方剂学杂志

Endophytic bacteria exist in the host and do not cause obvious damage to the host，which is an important part of the host ecosystem. Endophytic bacteria are a kind of important microbial resources with diverse species and wide distribution. In the process of long-term coevolution between endophytic bacteria and host，many symbiotic relationships，such as antagonism and reciprocity，have been gradually formed，which can directly or indirectly regulate the growth and development of host，assist host to resist diseases and insect pests，environmental stress and other biological processes. Endophytic bacteria can produce a variety of secondary metabolites in the process of symbiosis with the host，which plays an important role in the development of new natural products. In recent years，endophytic bacteria and their secondary metabolites have been widely used in the research and development of new drugs，biological control and other fields. In this paper，the diversity，species identification and secondary metabolites of endophytic bacterial resources are reviewed，and the future development direction is discussed，hoping to provide reference for the further development and utilization of endophytic bacterial resources.

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.

Analysis of genomic SSRs in Pueraria lobata and P. thomsonii and establishment of DNA identity card for different germplasms of P. thomsonii of Jiangxi province / 中国中药杂志

The type and frequency of simple sequence repeats( SSRs) in the genomes was investigated using the DNA sequence data of Pueraria lobata and P. thomsonii. Based on these SSRs,20 pairs of SSR primers were designed and 5 high polymorphism primer pairs were selected to analyze genetic diversity of 9 cultivars of P. thomsonii in Jiangxi province. The results showed that the 5 pairs of primers could generate 16 polymorphic alleles bands. The average polymorphism information content( PIC) of each SSR primer pair was 0. 600 7.According to the genetic similarity coefficients,the 9 cultivars of P. thomsonii can be classified into 6 germplasms. This study established DNA identity cards with 5 pairs of SSR primers for different germplasm resources of P. thomsonii in Jiangxi province,which provided reference information for the selection of fine germplasms of P. thomsonii and the theoretical basis for the study of Dao-di herbs.

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.

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.

Expression analysis of Hsp70-related miRNA in Lonicera japonica Thunb. during different flowering stages / 药学学报

Plant flowering regulation is an important mechanism to response to environmental stress. Heat shock protein 70 family is one of the main molecular chaperones to resist stress; miRNA can be used as a negative regulator to participate in post-transcriptional gene in flowering network. In this paper, we obtained an Hsp70 gene from Lonicera japonica transcriptome and combined with Lonicera japonica miRNA library to obtain a novel miRNA that may target Hsp70 gene through bioinformatics method. Bioinformatics and expression during different flowering stages of the obtained Hsp70 gene and miRNA were analyzed. Phylogenetic tree showed that the obtained Hsp70 gene was clustered with Hsp110 subfamily in Oryza sativa and Arabidopis thaliana. The prediction of miRNA secondary structure showed its stable structure and high reliability. The binding site map showed that there were two base mismatches between sequences of miRNA and Hsp70 gene. The expression analysis showed that the expression of Hsp70 and miRNA in different flowering stages had opposite trends, indicating that miRNA might regulate Hsp70 to participate in the flowering stages of Lonicera japonica. This study provided new ideas for Lonicera japonica flowering regulation and response to environmental stress mechanisms.

Establishment of DNA identity card and analysis of genetic similarity among 58 varieties in Lonicera japonica / 中国中药杂志

A total of 58 varieties in Lonicera japonica from 20 producing areas were amplified by 22 pairs of SSR primers. Seven pairs of polymorphic primers were screened and their primers were used to establish DNA identity card and analyze genetic similarity.All the 58 varieties could be distinguished each other by the DNA identity card constituted by 7 pairs of core SSR primers.The genetic similarity coefficients of 58 varieties ranged from 0.366 7 to 0.916 7 by using PopGene32（vesion1.32）. Furthermore, all the varieties consistency were classified into 4 groups and constructed an evaluation table according to cluster analysis by an un-weighted pair-group average method with arithmetic mean. As expected, the results of cluster and evaluation table reflected 58 varieties relatives, which provide reference information for the selection of fine germplasm of L. japonica and the theoretical basis for the study of Dao-di herbs.