[Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus].

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Isolation and Functional Characterization of New Terpene Synthase Genes from Traditional Edible Plants.

Terpene synthase (TPS) genes were isolated and functionally characterized from three traditional edible plants, Acanthopanax sciadophylloides ("Koshiabura") and Acanthopanax sieboldianus ("Himeukogi"), belonging to the family Araliaceae, and Curcuma zedoaria (zedoary, "Gajutsu"), belonging to the family Zingiberaceae. These plants emit characteristic fragrances and are used for traditional foods and folk medicines. From their fragrant tissues, i.e., sprouts of Araliaceae plants and developing rhizomes of zedoary, total RNAs were extracted and reverse transcribed. The resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. From the contig sequences obtained, full-length Tps genes were amplified by PCR with newly synthesized primer sets. The isolated full-length genes were introduced into engineered Escherichia coli cells, which can utilize acetoacetate to synthesize farnesyl diphosphate, the substrate for TPSs, through the mevalonate pathway. TPS products synthesized in the transformed E. coli cells were analysed by gas chromatography-mass spectrometry, nuclear magnetic resonance, and optical rotation. Consequently, the isolated Tps genes were found to encode ß-caryophyllene synthase, germacrene D synthase, linalool/(3S)-(+)-nerolidol synthase, ß-eudesmol synthase, and germacrene B synthase. These results lead us to expect that some of the effective ingredients in folk medicines are volatile terpenes and that intake of traditional foods including these edible plants would have some positive effects on our health.

Authentication of Eleutherococcus and Rhodiola herbal supplement products in the United Kingdom.

Siberian ginseng (Eleutherococcus senticosus, Araliaceae) and roseroot (Rhodiola rosea, Rosaceae) are popular herbal supplements which have been shown to improve resilience to conditions such as stress and exhaustion. Using DNA barcoding methods we tested 25 Siberian ginseng and 14 roseroot products which are widely available to UK customers to test whether the herbal ingredient stated on the label is also in the product. All Siberian ginseng supplements contained E. senticosus, however, 36% also contained an Eleutherococcus species other than E. senticosus. In three out of the 13 roseroot products which produced amplifiable DNA, we could only retrieve sequences matching alfalfa (declared on the product label) and fenugreek (not declared). In the other 10 supplements Rhodiola was detected but only five matched the target species R. rosea. As DNA can get severely degraded during the manufacturing process we did not take the absence of Rhodiola DNA as proof for a compromised product. Contamination could explain the presence of non-target species such as fenugreek but is unlikely to be account for the detection of congeneric Rhodiola species in roseroot preparations. Our results therefore suggest that the substitution or mixing of the target medicinal ingredient in these two popular supplements with other species is common.

The Identification of Araliaceae Species by ITS2 Genetic Barcoding and Pollen Morphology.

The genetic barcode ITS2 (ITS: internal transcribed spacer) and pollen morphology were used for the identification of the pharmacologically valuable wild Araliaceae species Panax ginseng, Oplopanax elatus, Aralia elata, Aralia continentalis, Eleutherococcus senticosus, and Eleutherococcus sessiliflorus inhabiting the natural forests of Primorye, Russia. The ITS2 locus successfully identified all six species, which supports the use of ITS2 as a standard barcode for medicinal plants. However, the ITS2 locus was insufficient for intra-specific discrimination in these species, neither within Primorye nor from other world representatives within GenBank. Araliaceae pollen was confirmed to undergo size-reducing metamorphosis. The final morphotypes were species-specific for each of the six species but could not discriminate intra-species geographic localities within Primorye. The morphologies of the final pollen morphotypes from homologous species inhabiting other parts of the world are not yet known. Therefore, whether pollen is applicable for Araliaceae intra-species discrimination between Primorye and other world localities could not be established. Based on these findings, we propose that the ITS2 genetic barcode and the final pollen morphotypes are suitable for the identification of Araliaceae species. However, further studies will be needed to determine the suitability of genetic and pollen traits for Araliaceae geographic authentication.

The complete chloroplast genome of Eleutherococcus gracilistylus (W.W.Sm.) S.Y.Hu (Araliaceae).

Eleutherococcus gracilistylus is a plant species that is close to E. senticosus, a famous medicinal plant called Siberian ginseng. The complete chloroplast genome sequence of the E. gracilistylus was determined by de novo assembly using whole genome next generation sequences. The chloroplast genome of E. gracilistylus was 156 770 bp long and showed distinct four partite structures such as a large single copy region of 86 729 bp, a small single copy region of 18 175 bp, and a pair of inverted repeat regions of 25 933 bp. The overall GC contents of the genome sequence were 36.8%. The chloroplast genome of E. gracilistylus contains 79 protein-coding sequences, 30 tRNA genes, and four rRNA genes. The phylogenetic analysis with the reported chloroplast genomes confirmed close taxonomical relationship of E. gracilistylus with E. senticosus.

Transcriptome profiling and digital gene expression by deep sequencing in early somatic embryogenesis of endangered medicinal Eleutherococcus senticosus Maxim.

Somatic embryogenesis (SE) has been studied as a model system to understand molecular events in physiology, biochemistry, and cytology during plant embryo development. In particular, it is exceedingly difficult to access the morphological and early regulatory events in zygotic embryos. To understand the molecular mechanisms regulating early SE in Eleutherococcus senticosus Maxim., we used high-throughput RNA-Seq technology to investigate its transcriptome. We obtained 58,327,688 reads, which were assembled into 75,803 unique unigenes. To better understand their functions, the unigenes were annotated using the Clusters of Orthologous Groups, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases. Digital gene expression libraries revealed differences in gene expression profiles at different developmental stages (embryogenic callus, yellow embryogenic callus, global embryo). We obtained a sequencing depth of >5.6 million tags per sample and identified many differentially expressed genes at various stages of SE. The initiation of SE affected gene expression in many KEGG pathways, but predominantly that in metabolic pathways, biosynthesis of secondary metabolites, and plant hormone signal transduction. This information on the changes in the multiple pathways related to SE induction in E. senticosus Maxim. embryogenic tissue will contribute to a more comprehensive understanding of the mechanisms involved in early SE. Additionally, the differentially expressed genes may act as molecular markers and could play very important roles in the early stage of SE. The results are a comprehensive molecular biology resource for investigating SE of E. senticosus Maxim.

[Establishment of prokaryotic expression and optimization ox expression conditions of Eleutherococcus senticosus P450 gene].

According to the sequence of P450 cDNA of Eleutherococcus senticosus, specific primers were designed. Frokaryotic ex pression vector pET30a-P450 was constructed and the prokaryotic expression conditions were optimized. Results showed that the BL21 after being transformed with the recombinant expression vector accumulated the high amount of recombinant protein. SDS-PAGE analysis showed that the recombinant protein was about 53 kDa. The recombinant accumulated the highest amount of recombinant protein af ter IPTG (1 mmol x L(-1)) at 27-37 degrees C for 24 h. Consequently P450 gene of E. senticosus could be expressed successfully by prokaryotic expression vector pET30a-P450. Induction temperature, IPTG concentration, medium type and amount of induction time could all influence the expression of target protein, but the impact strength was different.

[Expression profiles analysis of two member of squaleneepoxidase gene family from Eleutherococcus senticosus].

In order to find the characteristics of two members of gene family of squaleneexpoxidase (SE) , a quantitative real time PCR method was developed to analyze the expression of Eleutherococcus senticosus SE1 and SE2 gene from different growth periods and in different organs. The result indicated that all the expression of SE2 more than SE1 in the whole growth period and organs of E. senticosus. And in the whole growth period, expression of SE1 showed a low-high-low characteristic. Both expression of SE2 and growth period showed the same trend. The lowest content of the expression was in the roots. SE1 expression have been improved more than SE2 when treated with MeJA. The expression of E. senticosus SE1 and saponins content had significantly positive correlation (P < 0.05) and the correlation coefficients was 0. 858, while the correlation was not significant for SE2. That indicated that SE1 played a key enzyme gene in the biosynthesis of triterpenoidsaponins

[Genetic variation of the relict species Acanthopanax sessiliflorus (Rupr. et Maxim.) Seem. (Araliaceae) in Primorsky Krai].

Based on the analysis of 17 genes encoding the allozyme diversity of 12 enzyme systems, data were obtained on the genetic variation of a relict of the Tertiary flora, a valuable medicinal plant Acanthopanax sessiliflorus (Rupr. et Maxim.) Seem. (Araliaceae) in the Russian area of its habitat. Indicators of polymorphism for populations had rather high values on average (P95 = 42.4%, A = 1.55, H(o) = 0.211, and H(e) = 0.168), which are comparable with the known data for populations of A. sessiliflorus from the peninsula of Korea. The level of genetic diversity and its distribution among populations reflects the interaction of several factors, among which the most important are the historical past of the species, genetic drift, and the plasticity of the reproduction system. The obtained data can serve as a basis for the conservation of genetic resources of Far Eastern Araliaceae species.

[Analysis on codon usage of chloroplast genome of Eleutherococcus senticosus].

OBJECTIVE: To analyze the codon usage of chloroplast genome and the influencing factor in Eleutherococcus senticosus. METHOD: Codon of 52 genes, which were selected from the chloroplast genome sequence of E. senticosus, was multivariate statistical and correspondence analyzed using CodonW and SPSS software. RESULT: GC content at the three position of codons by turns was 46.46%, 38.26%, 29.88%, whereas GC1 and GC2 had a significant correlation coefficient (P < 0.01). The correlation coefficient with GC12, and GC3 was 0.205 and was not significant correlated. There were 30 codons which relative synonymous codon usage was greater than 1 and 29 codons end with A and T. In the corresponding analysis, the first axis shows 10.35% variation. And there was significant correlation coefficient between ENC and GC3. The correlation coefficients with GC3 and ENC were -0.288 and 0.353, respectively. We defined 16 codons from 16 amino acids as the major preference codons in chloroplast genome of E. senticosus. CONCLUSION: The third positions for all codon are preferred to ending with A and T. The codon usage bias is formed under effect of mutation and selection, as well as other factors. But the selection will have a far greater impact than others.

[Cloning of Eleutherococcus senticosus calmodulin gene and effect of endophytic fungus on expression amount of gene].

OBJECTIVE: To clone calmodulin (CaM) gene in Eleutherococcus senticosus, and study the effect of endophytic fungi on expression amount of CaM gene. METHOD: The CaM full length cDNA sequence was cloned by rapid amplification of cDNA ends (RACE). The gene was analyzed and corresponding structure and functions were predicted by the bioinformatics methods. The expression amount of CaM gene affected of endophytic fungus P116-1a, P116-1b, P1094 and P312-1 was detected by RT-PCR. RESULT: The full length of CaM cDNA was 856 bp containing an ORF of 450 bp that encoded a protein of 149 amino acids. The homologous of predicted protein was almost 100% with plants like Panax ginseng and Daucus carota. RT-PCR results showed that endophytic fungus improved CaM expression amount significantly (P<0.05). The highest expression amount of CaM occurred 90 d after reinoculated with endophytic fungi P1094, up to 2.96 times of the control. CONCLUSION: The CaM gene of E. senticosus was successfully cloned for the first time. The results demonstrated that endophytic fungus of E. senticosus improved CaM expression amount significantly.

[Molecular cloning of farnesyl diphosphate synthase from Eleutherococcus senticosus and its bioinformatics and expression analysis].

OBJECTIVE: To clone farnesyl diphosphate synthase (FPS) gene from Eleutherococcus senticosus and analyze the bioinformatics and expression of the gene. METHOD: The FPS full length cDNA was cloned by rapid amplification of cDNA ends (RACE). The data was analyzed by bioinformatics method, the structure and function of FPS was deduced. The expression of FPS in different organ of E. senticosus was detected by RT-PCR. RESULT: The full length of FPS cDNA was 1 499 bp containing a 1 029 bp ORF that encoded 342 amino acids. The deduced protein sequence exhibited two Asp riches conserved motifs (DDXXD). Without transmembrane domain, FPS was located in cytoplasm. RT-PCR result showed that FPS gene expressed in different organs of E. senticosus. The expression amounts of FPS in different organs were different significantly (P < 0.05). CONCLUSION: The FPS gene of E. senticosus was successfully cloned for the first time, and provided a stable foundation for studying on its effect and expression control on E. senticosus saponins biosynthesis.

[Cloning and sequence analysis on cDNA of squalene epoxidase gene in Eleutherococcus senticosus].

OBJECTIVE: To clone and sequence the cDNA of squalene epoxidase gene in Eleutherococcus senticosus. METHOD: Total RNA of E. senticosus was extracted by the improved isothiocyanate method and reverse transcripted into cDNA. The primers were designed depending on the reported SE cDNA sequences of Panax ginseng. The SE cDNAs in E. senticosus was amplified using RT-PCR strategy. RESULT: Sequencing results showed two different cDNA fragments (SE1, SE2) with 1665, 1629 bp each ORF which encoded 554,542 amino acids, respectively. The identities of nucleotides and amino acids between SE1, SE2 were 91.49%, 92.55%. SE1, SE2 had the highest amino acids similarity to the SE1 of P. notoginseng, 93.45%, 94.87% respectively. SE1, SE2 both had a FAD binding domain. The deduced speculated amino acids of SE1, SE2 each had 2,4 membrane-spanning helices. CONCLUSION: The two SE sequences in E. senticosus were firstly separated and reported, which has made foundation for E. senticosus secondary metabolite engineering researches.

Extract from Acanthopanax senticosus harms (Siberian ginseng) activates NTS and SON/PVN in the rat brain.

The extract of the stem bark of Siberian ginseng, Acanthopanax senticosus Harms (ASH), is believed to play a body-coping role in stress through a brain noradrenergic mechanism. The present study was carried out to investigate the effect of ASH on the neuronal activation patterns of c-Fos expression in the rat brain. With ASH administration, c-Fos accumulated in both the supraoptic nuclei (SON) and paraventricular nuclei (PVN), which regulate stress response. Only the caudal regions in the nucleus of the solitary tract (NTS), a locus innervating both the SON and PVN, were activated. Such a neuro-anatomical pattern associated with ASH suggests the possible involvement of these stress-related brain loci.

Authentication of the traditional medicinal plant Eleutherococcus senticosus by DNA and chemical analyses.

Shigoka (SGK), the rhizome of Eleutherococcus senticosus, is a traditional medicine used as a tonic in northeastern Asia and far eastern Russia. We analyzed the nuclear ribosomal DNA internal transcribed spacer (ITS) sequence of the medicine available on the Japanese and Chinese markets and found that at least 3 species were used as the source plant of the commercial SGKs and that only 70% of all samples was made from the correct species. Furthermore, we performed the quantitative determination of 3 marker compounds, eleutheroside B (EB), syringaresinol diglucoside (Syr), and isofraxidin (Iso) by ultraperformance liquid chromatography (UPLC)/mass spectrometry (MS). We found that EB and Iso are specific to the correct source plant of SGK. Of them, EB is thought to be the best marker compound for quality assurance of the SGK from the viewpoint of its pharmacological activity.

Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng.

The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.

Intraspecific relationship analysis by DNA markers and in vitro cytotoxic and antioxidant activity in Eleutherococcus senticosus.

To analyse genetic relationships and intraspecific variation within Eleutherococcus senticosus, the polymerase chain reaction (PCR) was performed on total genomic DNAs of 10 Eleutherococcus collections. Ten primers were used for amplification, yielding 106 bands, of which 87 were polymorphic. The genetic diversity and genetic distance among 10 collections of Eleutherococcus species were used to describe the dendrogram showing the phylogenic relationship. The 10 collections were classified into two groups (groups I and II) at a similarity coefficient of 0.50. Group I included E. senticosus from Bukhaedo (Japan), E. sessliliflorus from Youngwal (Korea), E. seoulense and E. chiisanesis, while group II included several internal and Russian collections. The range of polymorphism was from 66.7 to 90.9% in the 87 amplified polymorphic DNA fragments. The similarity value of all collections ranged from 0.41 to 0.92, and the average genetic distance was 0.61. Thus, RAPD analysis was useful in determining genetic relatedness among collections and in identifying different genotypes of E. senticosus and other Eleutherococcus species. Also, the biological activity on DPPH radical scavenging, antilipid peroxidation in rat liver microsomes and cytotoxic sulforhodamine B (SRB) assay was evaluated using root extracts of E. senticosus, Odaesan, Korea. Ethyl acetate and n-butanol fractionation revealed strong antioxidant against scavenging on DPPH free radical and also ethyl acetate fractionation exhibited high antilipid peroxidative activities. In the cytotoxic effects were evaluated on seven human cancer cell lines, the values of 50% growth inhibition (GI(50)) were mostly below 30 microg/ml for crude extracts to be considered as significantly active.