Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus / 中国中药杂志

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.

Effects of fungi fraction on growth and anti-oxidative activity of Eleutherococcus senticosus / 中国中药杂志

The present study was conducted to explore the effect of endophytic fungi fraction on growth and anti-oxidative activity of Eleutherococcus senticosus. The growth,yield,contents of MDA,and antioxidant activities were assessed in E. senticosus under five fungi fractions,namely BZ,MH,DT,JS,and XFZ. The results showed that fungi fractions and component significantly affected the growth,low concentration of DT fungi fraction significantly increased the biomass of E. senticosus,reduced the MDA content in cells,and the antioxidant activities of the aqueous extracts were superior to the others. The results indicated that low concentration of DT fungi fraction was the optimum fraction to achieve high yield and quality of E. senticosus.

Cloning and expressive characteristics of GPS gene and its correlation with saponins content in Eleutherococcus senticosus / 中草药

Objective To clone cDNA sequence of geranyl pyrophosphate synthase (GPS) gene from Eleutherococcus senticosus and analyze genetic characteristics, gene expression level in different organs and the correlation between GPS gene expression and saponins content. Methods RNA was extracted from E. senticosus and reverse transcribed into cDNA. Gene specific primers were designed according to the unigene (c37362.graph_c0) of GPS from transcriptome sequencing data. The full length of the GPS gene cDNA was amplified by PCR. The expression level of GPS gene in different organs was analyzed by qRT-PCR. The content of E. senticosus saponins was detected by spectrophotometry method. Results GPS gene cDNA was cloned from E. senticosus and encodes 419 amino acids with full length of 1 260 bp. GPS protein located in mitochondria does not have transmembrane region. The GPS gene was expressed in each organ and had the highest expression in blade, which is 5.26 times in root. The relative expression of GPS gene and content of saponin showed the same trend and significant positive correlation (r = 0.851, P < 0.05). Conclusion The whole length of cDNA sequence of GPS gene is cloned for the first time, and there is a positive correlation between the expression level of GPS gene and the saponin content of E. senticosus.

Prediction of CpG islands and functional verification on promoters of FPS, SS, SE in Eleutherococcus senticosus / 中草药

Objective: To obtain the distribution and functional activity of CpG islands in promoters of FPS, SS, and SE from Eleutherococcus Senticosus. Methods: Based on the promoter sequence of FPS, SS and SE, CpG islands were predicted by using EMBOSS and Li Lab. The functional verification of transformed Arabidopsis thaliana was mediated by Agrobacterium tumefaciens by using GUS and pCAMBIA1301 plasmid as the reporter gene and expression vector respectively. Results: Two CpG islands were found in FPS and SS promoter with the lengths of 520 bp, 218 bp and 108 bp, 103 bp, and three CpG islands in SE promoter in 290 bp, 119 bp and 149bp. The promoters of FPS, SS and SE all had promoter activities at different level, in which SS promoter was the highest one. Conclusion: The functional verification and distributions of CpG islands in the promoters area of FPS, SS, and SE were reported in this research at first time, which established the foundation for the methylation analysis of FPS, SS, and SE and the further studying of the mechanism expression regulation in E. Senticosus.

Analysis on structure and phylogeny of chloroplast genomes in Araliaceae species / 中草药

Objective: To obtain the structural characteristics and phylogentic relationships of the chloroplast genome in Araliaceae species. Methods: we used 20 chloroplast genomes which have sequenced as materials, they were from 20 species belonging to 10 genus of Araliaceae. Analysis the differences of genomes and the dilation or shrink of four boundaries for IR, we used MEGA 4.0 to build the phylogenetic tree with Angelica gigas of sibling species as the outgroup and analysis their phylogentic relationships. Results: There was a small difference among the chloroplast genomes size, and the largest difference is 1 909 bp. All of species had existed gene replacements, cemA replaced ycf10, and number of genes existed some differences, they were mainly caused by tRNA. The four boundaries of IR was relatively conservative, only Panax vietnamensi, Panax notoginseng and Schefflera delavayi were special, their boundary genes were in IR. All nodes of the phylogenetic tree of Araliaceae which was based on Angelica gigas were of high supports, and the tree had good resolution to reflect the genetic relationships among Araliaceae. Conclusion: Chloroplast genomes have a lot of information, it can be used to analysis phylogeny among the species which are affinity or faster evolution.

Composition, antioxidant and antimicrobial activities of Eleutherococcus senticosus fruit extracts.

This study was designed to examine the in vitro antioxidant and antimicrobial activities of essential oil and crude methanolic (MeOH) extract of Eleutherococcus senticosus (common name, Siberian ginseng) fruit and its partitioned fractions, including hexane, ethyl acetate (EtOAc), n-butanol (BuOH) and aqueous. 1,1-dephenyl-2-picryl-hydrazyl free radical scavenging and reducing power assay suggest that antioxidant activity of EtOAc and BuOH fractions is due to the reducing ability of the antioxidant against oxidative effects of reactive oxygen species. In addition, the essential oil of Siberian ginseng fruit showed remarkable antimicrobial activity against Kocuria rhizophila (MIC = 125 μg/ml), Micrococcus luteus (MIC = 500 μg/ml), and Escherichia coli (MIC = 63 μg/ml). The chemical compositions of the essential oil obtained by the simultaneous steam-distillation and extraction method were analyzed using GC-MS; trans-caryophyllene (21.7%), humulene (7.4%), bicyclogermacrene (6.0%), (+) spathulenol (4.5%), germacrene-D (3.2%), tau-muurolol (2.5%) and delta-cadinene (2.3%) were found as main constituents. Furthermore, HPLC analysis identified ursolic acid as one of the principal components in Siberian ginseng fruit extract. Although this study has been carried out by in vitro assay, these results suggest that Siberian ginseng fruit may be a good candidate as a source of antioxidant and antimicrobial ingredients.

Cloning and analysis on GAPDH genomic DNA from Eleutherococcus senticosus / 中草药

Objective: To clone and analyze the full length DNA and promoter sequence of GAPDH gene from Eleutherococcus senticosus. Methods: PCR and TAIL-PCR techniques were used to clone the full length DNA sequence and promoter sequence of GAPDH gene of E. senticosus, then these two sequences were analyzed by bioinformatics methods. Results: Length of 4 103 bp of E. senticosus GAPDH gene DNA and promoter sequence was cloned. Gene structure analysis showed that it contained 12 exons and 11 introns, and the splicing principles of its exon and intron were consistent with GT-AG; The promoter sequence length was 1 304 bp, and the transcription start site located 61 bp upstream of the initiation codon ATG; The promoter elements such as TATA-box, CAAT-box, as well as many cis-regulatory elements were related to hormone signal response, light response and stress signals. Conclusion: The full length DNA and promoter sequence of GAPDH gene in E. senticosus is successfully cloned and reported for the first time, and it provides a stable foundation for further study of GAPDH gene structure and function of E. senticosus.

Cloning and bioinformatic analysis of mevalonate diphosphate decarboxylase gene promoter in Eleutherococcus senticosus / 中草药

Objective: To clone and analyze the sequence of mevalonate diphosphate decarboxylase (MDD) gene promoter from Eleutherococcus senticosus. Methods: According to the full length of MDD cDNA sequence, using PCR cloning and TAIL-PCR methods, we are cloning 5'DNA sequence and promoter sequence of MDD gene. And bioinformatic analysis is used. Such as PlantCARE. Results: We have cloned the 5'DNA sequence successfully, with full length of 1024 bp. And the promoter sequence is 1423 bp. Bioinformatic analysis shows that the promoter sequence has 49 TATA-box and 25 CAAT-box. In addition, the promoter has some cis-acting elements responsive to abscisic acid, environmental stresses, MeJA, required for endosperm expression, light-regulated and so on. Last but not least, there have two MYBHv1 and two MYB binding sites. Conclusion: We have firstly cloned and analyzed the E. senticosus MDD gene promoter sequence. It is important to it's expression.

Analysis of transicriptomes and differentially expressed genes in Eleutherococcus senticosus / 中草药

Objective: To obtain the transcriptome database and differentially expressed genes of Eleutherococcus senticosus. Methods: We choose the high content group and the low content group of saponin as experimental materials, and use the high-throughput sequencing technology (Illumina HiSeq 4000) to sequence the transcriptome of E. senticosus, then we systematically analyze the sequencing results in the bioinformatic way. Results: We have assembled 8.34 Gb database, after assembly steps, we get 77 087 of E. senticosus unigenes, then blasting them with five data banks. All unigenes are involved in 55 GO-terms and 116 metabolic pathways. Though the differentially expressed analysis of two materials, we get 530 differentially expressed genes, the up-regulated genes account for 42.08%, the down-regulated genes account for 57.92%. After GO and Pathway enrichment analysis, we get 408 GO-natations and 40 metabolic pathways. Conclusion: These data represent the abundant messeges about transcripts and provide the valuable genome data sources in molecular biology of E. senticosus.

Gene cloning and expression level of bAS and their correlation with content of saponins in Eleutherococcus senticosus / 中草药

Objective: To clone β-amyrin synthase (bAS) gene from Eleutherococcus senticosus and analyze the effect of its expression on saponin contents. Methods: The sequence of cDNA of E. senticosus bAS was cloned by homologous cloning strategy. Quantitative real time PCR was developed to analyze the expression pattern of E. senticosus bAS gene. And E. senticosus saponin contents were measured by spectrophotometry method. Results: Length 1 223 and 1 226 bp of E. senticosus bAS1 and bAS2 genes were cloned. The results showed that bAS1 and bAS2 were expressed in the each growth period and every organ of E. senticosus, and the expression differed significantly (P < 0.05). bAS1 showed the highest expression when the plants were grown in germination stage, then rapidly depressed, and changed slightly in the end. The expression of bAS2 showed the characteristic of low-high-low. The expression of bAS1 in different organs of E. senticosus was constant, but the highest content of the expression of bAS2 was in the leaves. With the treatment of methyl jasmonate (MeJA), bAS2 expression has been significantly improved and bAS1 without a significant changing. There exists significantly positive correlation (P < 0.01) between the content of E. senticosus saponins and the expression levels of bAS2, and bAS1 without a significant difference. Conclusion: bAS2 may be a key enzyme gene in the biosynthesis of triterpenoid saponins.

Clone of Full-length cDNA Sequence of Actin Gene of Eleutherococcus Senticosus and Bioinformatics Analysis / 世界科学技术-中医药现代化

This study was aimed to clone the full-length cDNA sequence of actin gene of Eleutherococcus sentico-sus. And bioinformatics analysis was used. The total RNA was isolated from leaves of E. senticosus , and cDNA was synthesized by reverse transcription of total RNA. Primers were designed according to the conserved se-quence that had been cloned of Actin of E. senticosus . Then, the 3'and 5' cDNA fragments were cloned by nested PCR . The full-length gene was obtained by gene splicing method . Sequencing results were compared and treated with similarity analysis by blast analysis in the GenBank. Protein secondary structure and tertiary struc-ture of Actin of E. senticosus was predicted by online software. The results showed that the full-length cDNA of Actin of E. senticosus is 1507 bp, which named EsActin1, GenBank accession No. KC469585. The conserved sequence, which contained a 1134 bp open reading frame that encoding a 377 amino acid residues, a 5'-UTR of 140 bp and a 3'-UTR of 233 bp. Homologous alignment showed that it shared over 75% nucleotide se-quence similarity and over 94% amino acid sequences similarity with Actins in other plants. It was concluded that this study first isolated and reported the full-length cDNA sequence of actin gene of E. senticosus , and laid a foundation for the molecular biology research of E. senticosus .

Analysis on bias of functional gene codon of Eleutherococcus senticosus / 中草药

Objective: To analyze the codon usage of functional genes in Eleutherococcus senticosus and their influencing factors. Methods: The multivariate statistical analysis and correspondence analysis were carried out using CodonW and SPSS software with codon of 17 genes selected from the functional genes of E. senticosus. Results: GC contents at the three positions of functional gene codons in E. senticosus was 51.03%, 41.23%, and 40.04%, all of which had a significant correlation coefficient (P < 0.05). The correlation coefficients with GC12 and GC3 were 0.262, and both were insignificant. The relative synonymous codon usage of 27 codons was greater than 1, among which 22 codons ended with A or T base. In the corresponding analysis, the first axis showed the variation of 22.78%, and there were significant correlation coefficients in codon adaptation, codon bias index, and GC3 (P < 0.01). The correlation coefficients were 0.686, 0.617, and 0.786, respectively, but they were insignificantly related with the effective number of codons (ENC). The second axis showed the variation of 19.28% and it was only significantly related with ENC (r = 0.635). Seventeen optimized codons in functional genes of E. senticosus were defined. Conclusion: All codons of functional genes in E. senticosus prefer to ending with A or T base. The codon usage bias is formed under the effects of mutation and selection.

Cloning and expression stability analysis of actin gene in Eleutherococcus senticosus / 中草药

Objective: To clone the actin (ACT) gene of Eleutherococcus senticosus, and to make the gene a valuable internal gene. Methods: Part of the sequence of ACT gene was cloned by real-time PCR (RT-PCR), and the sequence was used as internal control gene for analyses of semiquantitative PCR and RT-PCR. Results: The ACT gene (1031 bp) of E. senticosus was cloned, coding 343 amino acids. To compare the amino acid sequence of E. senticosus ACT gene with those of Betula luminifera, Gossypium hirsutum, and Camellia sinensis, the amino acid homology was 99.42%, 98.83%, and 98.54%. The expression of ACT in different organs of E. senticosus during various growing periods was constant. The expression of ACT gene in different organs and during different growth and development stages was basically constant, and when the sequence acted as internal control gene, the semiquantative PCR and RT-PCR have good amplification effect and reproducibility. Conclusion: The ACT sequence in E. senticosus is firstly separated and reported, it could act as an internal control gene, and its reaction system of RT-PCR is established.

Cloning and sequence analyses of Eleutherococcus senticosus GAPDH / 中草药

Objective In order to make the gene a valuable internal control gene, GAPDH of Eleutherococcus senticosus was cloned and analyzed in sequence. Methods Total RNA of E. senticosus was extracted using improved isothiocyanate method. Part sequence of GAPDH was coined by RT-PCR, and the sequence was acted as internal control gene for semiquantitative PCR. Results Length 627 bp of E. senticosus GAPDH was cloned, speculating coding 209 amino acids. To compare the amino acid sequence of £. senticosus GAPDH with those of Panax notoginseng, P. ginseng, and Arabidopsis thaliana, the amino acid homology was 97%, 93%, and 93%, respectively, and the nucleotide homology was 94%, 86%, and 84%, respectively. When the sequence acted as internal control gene, the semiquantative PCR has benign amplification effect and good reproducibility. Conclusion The cDNA clone of E. senticosus GAPDH is first reported, the results prove that the sequence is able to be internal control gene for analysis on gene expression. This study could make a foundation for the key enzyme expression and regulate mechanism analysis in eleutheroside biosynthesis pathway.

Immune Cell Activation and Co-X-irradiation Effect of Eleutherococcus senticosus Maxim Root / 대한방사선종양학회지

PURPOSE: This study was performed to investigate the effects of immune cell activation and the antitumor effect for the combination of treatment with X-irradiation and Eleutherococcus senticosus Maxim Root (ESMR) on mouse tumor cells. MATERIALS AND METHODS: ESMR (250g) was extracted with 80% methanol, concentrated under decompression and lyophilized. To determine whether ESMR is able to activate the immune cells or not, the proliferation of splenocytes in vitro and the number of B cells and T cells in splenic lymphocytes in ESMR-pretreated mice were evaluated. X-irradiation was given to the mouse fibrosarcoma tumor cells (FSa II) by 250 kv X-irradiation machine. The cytotoxicity of ESMR was evaluated from its ability to reduce the clonogenecity of FSa II cells. In X-irradiation alone group, each 2, 4, 6 and 8 Gy was given to FSa II cells. In X-irradiation with ESMR group, 0.2 mg/ml of ESMR was exposed to FSa II cells for 1 hour before X-irradiation. RESULTS: The proliferation of cultured mouse splenocytes and thymocytes were enhanced by the addition of ESMR in vitro. The number of B cells and T cells in mouse splenic lymphocytes was significantly increased in ESMR pretreated mice in vivo. In FSa II cells that received a combination of 0.2 mg/ml of ESMR with X-irradiation exposure, the survival fraction with a dose of 2, 4 and 6 Gy was 0.39+/-0.005, 0.22+/-0.005 and 0.06+/-0.007, respectively. For FSa II cells treated with X-irradiation alone, the survival fraction with a dose of 2, 4 and 6 Gy was 0.76+/-0.02, 0.47+/-0.008 and 0.37+/-0.01. The difference in the survival fraction of the mouse FSa II cells treated with and without ESMR was statistically significant (p<0.05). CONCLUSION: Treatment with ESMR increased cell viability of mouse splenocytes in vitro and especially the subpopulation of B cells and T cells in splenocytes in ESMR-pretreated mice. However, treatment with ESMR did not increase the level of Th and Tc subpopulations in the thymocytes. Treatment with the combination of ESMR and X-irradiation was more cytotoxic to mouse tumor cells than treatment with X-irradiation alone; this finding was statistically significant.

Somatic embryogenesis from petiole in Eleutherococcus senticosus / 中草药

Objective The petiole of Eleutherococcus senticosus was used as somatic embryo,which is widely original plants with the pharmacological active components whose contents could be determined,the somatic embryo in E.senticosus was studied,the aim of this study is to provide the proof of E.senticosus species which have the higher yield of pharmacological active components.Methods Using the petiole of E.senticosus of three years old plants germinated somatic embryos within 15 d to observe the somatic embryogenesis of E.senticosus with the 2,4-D+BA medium.Results After cultured for 28 d with 2,4-D 1.5 mg/L+BA 1.0 mg/L,71.4% of the petiole somatic embryos were directly produced or 8.5 embryos in total were produced via callus.Both of the two methods could be used in the elicitation medium,but the percentage of indirect production was smaller.After transforming into the same or the lower concentration of 2,4-D medium,the somatic embryos gradually matured.At the same time,those of the new somatic embryos were also produced,the percentage of the somatic embryos which were produced by indirect way was increased with it.Conclusion Using the petiole of E.senticosus germination within 15 d could make somatic embryogenesis.It confirms that the somatic embryogenesis and the bodybmeryos inductivity depend on the 2,4-D and BA concentration.