[Application of strigolactone analogs in storage of Gastrodia elata].

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.

[Effect of BBM gene on callus growth and ginsenoside content in Panax quinquefolius].

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.

[Screening of an Armillaria gallica strain for Gastrodia elata cultivation].

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.

Metabolic and Transcriptional Stress Memory in Sorbus pohuashanensis Suspension Cells Induced by Yeast Extract.

Plant stress memory can provide the benefits of enhanced protection against additional stress exposure. Here, we aimed to explore the responses of recurrent and non-recurrent yeast extract (YE) stresses in Sorbus pohuashanensis suspension cells (SPSCs) at metabolomics and transcriptional levels. Biochemical analyses showed that the cell wall integrity and antioxidation capacity of SPSCs in the pretreated group were evidently improved. Metabolic analysis showed that there were 39 significantly altered metabolites in the pretreated group compared to the non-pretreated group. Based on the transcriptome analysis, 219 differentially expressed genes were obtained, which were highly enriched in plant-pathogen interaction, circadian rhythm-plant, oxidative phosphorylation, and phenylpropanoid biosynthesis. Furthermore, the correlation analysis of the transcriptome and metabolome data revealed that phenylpropanoid biosynthesis involved in the production of biphenyl phytoalexins may play a critical role in the memory response of SPSC to YE, and the key memory genes were also identified, including PAL1, BIS1, and BIS3. Collectively, the above results demonstrated that the memory responses of SPSC to YE were significant in almost all levels, which would be helpful for better understanding the adaptation mechanisms of medicinal plants in response to biotic stress, and laid a biotechnological foundation to accumulate favorable antimicrobial drug candidates from plant suspension cells.

[Differences between male and female leaves of Schisandra sphenanthera: based on RNA-Seq].

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.

[Genetic diversity of protopine-6-hydroxylase in three medicinal Papaver plants].

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.

[Bioinformatics analysis and function prediction of ginseng TCP transcription factor family].

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.

[Rapid quality detection system of Lonicerae Japonicae Flos formula granules].

The application of traditional Chinese medicine(TCM) formula granules in clinical practice is gradually extensive. However, TCM formula granules is still lacking rapid and simple quality control standards. In this study, allele-specific PCR and enzyme-linked immunoassay(ELISA) was used for rapid detection of the quality of Lonicerae Japonicae Flos formula granules. The authenticity of Lonicerae Japonicae Flos formula granules was identified by allele-specific PCR and index component was detected by ELISA. Thus, it lays a foundation for the establishment of rapid quality detection standard for Lonicerae Japonicae Flos formula granules, and also provides reference for other studies on the quality standard of traditional Chinese medicine formula granules.

[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].

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].

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.

[Bioinformatics analysis and expression of chitinase genes in Armillaria gallica].

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 monosaccharide composition of twelve polysaccharides by pre-column derivatization procedure UPLC-MS/MS].

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.

[Establishment of DNA identity card and analysis of genetic similarity among 58 varieties in Lonicera japonica].

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.

[Correlative analysis advance of chemical constituents of Polyporus umbellatus and Armillaria mellea].

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.

Two new sesquiterpenes from Chloranthus japonicus Sieb.

Two new sesquiterpenes, namely, 1ß,10ß-dihydroxy-eremophil-7(11), 8-dien-12,8-olide (1) and 8,12-epoxy-1ß-hydroxyeudesm-3,7,11-trien-9-one (2), together with three known sesquiterpenoids, shizukolidol (3), 4α-hydroxy-5α(H)-8ß-methoxy-eudesm-7(11)-en-12,8-olide (4), and neolitacumone B (5), and two known monoterpenes, (3R,4S,6R)-p-menth-1-en-3,6-diol (6) and (R)-p-menth-1-en-4,7-diol (7), were isolated from the whole plant of Chloranthus japonicus Sieb. Their structures were elucidated on the basis of spectroscopic data analysis and comparison with those of related known compounds. Compounds 4-7 were isolated from this plant for the first time.

Three Sesquiterpenoid Dimers from Chloranthus japonicus: Absolute Configuration of Chlorahololide A and Related Compounds.

A novel sesquiterpenoid dimer, named multistalide C (1), together with two known congeners, shizukaols C (2) and D (3), was isolated from the whole plant of Chloranthus japonicus Sieb. The structures of compounds 1-3 were elucidated by extensive HR-ESI-MS, 1D, and 2D NMR spectroscopic analysis. Compounds 1-3 exhibited significant toxic effects on brine shrimp larvae (Artemia salina). The absolute configuration of 1 was established by CD/TDDFT calculations. The related compound chlorahololide A was also reinvestigated. The previous assignment of the absolute configuration of chlorahololide A and several related sesquiterpenoid dimers, based on an incorrect application of the exciton chirality method, is criticized.

[Experimental verification and prospect on stress effect of Dao-di herbs Scutellaria baicalensis].

The stress effect is a characteristic of Dao-di herbs caused by environmental gene expression of medical plants is influenced by environmental changes and finally affects the formation and accumulation of metabolites. Using Scutellaria baicalensis as material, active component of wild type of S. baicalensis from 19 production areas were analysed; It was found that climate change can influence the accumulation of active components. Then, S. baicalensis suspension cells was exposed to various environments, and enzyme activity and gene expression were measured, indicating the molecular mechanism of stress effect on S. baicalensis. Hence, we found the prerequisite and method to study the stress effect on Dao-di herbs, and we hope this research can provides some references for another studies of Dao-di herbs.

[Germplasm identification of Lonicerae Japonicae Flos by DNA melt curve analysis].

To establish a new high resolution melting analytical method for identification of Lonicera japonica germplasm, the screening of 7 pairs of SSR (simple sequence repeats) primers, determining the suitable diagnostic primers by the differences of peak pattern and Tm was conducted. Then into the DNA template concentration, annealing temperature and the suitable range of cycle number were investigated. Combined with SIMCA-P software for data processing analysis, the results show that three main germplasm honeysuckle could be divided by four sets of primers. It provides methodology for improving L. japonica germplasm identification.

[Effect of curcumin on caspase-12 and apoptosis in pulmonary ischemia/reperfusion injury mice].

OBJECTIVE: To explore the effect of curcumin (CUR) on cycteinyl aspirate specific protease-12 (Caspase-12) and pneumocyte apoptosis in pulmonary ischemia/reperfusion (I/R) injury mice. METHODS: The in vivo unilateral in situ pulmonary I/R injury mouse model was established in C57BL/6J mice. Sixty experimental mice were randomly divided into six groups by random digit table, i. e., the sham-operation group (Sham), the I/R group, the I/R + dimethyl sulfoxide group (I/R + DMSO), the I/R + low dose CUR pre-treated group (I/R + CUR-100), the I/R + middle dose CUR pre-treated group (I/R + CUR-150), the I/R + high dose CUR pre-treated group (I/R + CUR-200), 10 in each group. Mice were euthanized and their left lungs were excised. Wet lung weight to dry lung weight (W/D) and the total lung water content (TLW) were tested. The morphological changes of the lung tissue were observed and index of quantitative evaluation for alveolar damage (IQA) detected under light microscope. The ultra-microstructure of the lung tissue was observed under electron microscope. The mRNA and protein expression levels of Caspase-12 and glucose regulated protein (GRP78) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Apoptosis index (AI) of the lung tissue was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method. RESULTS: Compared with the Sham group, expression levels of Caspase-12, GRP78 mRNA and protein all significantly increased in the I/R group (P < 0.05); W/D, TLW, IQA, and AI were all notably higher (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were notably observed in I/R group. Compared with the I/R + DMSO group, expression levels of GRP78 mRNA and protein were increasingly higher in the I/R + CUR-100 group, the I/R + CUR-150 group, and the I/R +CUR-200 group (P < 0.05), expression levels of Caspase-12 mRNA and protein were lower (P < 0.05); W/D, TLW, IQA, and AI also decreased (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were gradually alleviated in the I/R + CUR groups. CONCLUSION: CUR had better effect on the lung protection against I/R injury, which might be related to inhibition for pneumocyte apoptosis associated with Caspase-12 in excessive unfolded protein response (UPR).

X-ray crystallographic study of ranaconitine.

The crystal structure of natural diterpenoid alkaloid ranaconitine isolated from Aconitum sinomontanum Nakai has been determined by single crystal X-ray diffraction analysis. The crystal presents a monoclinic system, space group C2 with Z = 4, unit cell dimensions a = 30.972(19) angstrom, b = 7.688(5) angstrom, and c = 19.632(12) angstrom. Moreover, the intermolecular O-H...O hydrogen bonds and weak pi-pi interactions play a critical role in expanding the dimensionality.