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As one kind of v-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factors, R1-MYB (MYB-related) family plays an important role in plant growth and development, as well as environmental stress and hormone signal transduction. In this study, R1-MYB family genes in Rheum palmatum L. were systematically screened based on full-length transcriptome sequencing analysis. Firstly, the physicochemical, protein domain and molecular evolution characteristics of the coding proteins were analyzed. Furthermore, the tissue expression levels of R1-MYB genes were analyzed by RNA-seq. We also investigated the expression pattern of RpMYB24 in response to various hormones and abiotic stresses. The results showed that a total of 49 R1-MYB genes were identified, which mainly encoded thermally stable hydrophilic proteins. Most of the deduced proteins were predicted to locate in nucleus. Each protein had a large proportion of random curl and α helix, and also had the W-type conserved amino acids which were the signature of MYB. R1-MYB family members were distributed in five subgroups, including circadian clock associated 1 (CCA1)-like, I-box (GATAAG)-like, CAPRICE (CPC)-like, telomere repeat binding factor (TRF)-like and TATA binding protein (TBP)-like, and the number of CCA1-like was the majority. RNA-seq revealed that 49 R1-MYB genes were differentially expressed in roots, rhizomes and leaves of R. palmatum, and the expression levels of 15 and 23 genes in roots and rhizomes were higher than those in leaves, respectively. RpMYB24 transcript was induced by abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) treatment, and could also significantly respond to injury, low temperature and high temperature stresses except drought stress. This study systematically identified the R1-MYB family genes and their molecular characteristics, better for further gene functional validation, and then provide a scientific basis for the transcriptional regulation mechanism research into rhubarb quality formation.
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MYB transcription factors play many important regulatory roles in plant growth and development, secondary metabolism, and stress adaptation processes. In this work, an MYB gene containing a complete open reading frame (ORF) was selected from the transcriptome database of R. palmatum L. RpMYB4 ORF and cloned, encoding a polypeptide of 245 amino acids with a molecular weight of 26.99 kDa. RpMYB4 lacks a signal peptide or transmembrane domain but contains two conserved DNA binding domains (HTH-MYB) of the R2R3-MYB subfamily at the N-terminus. Multiple-sequence alignment demonstrated that RpMYB4 shared as high as 61% identity with many MYB proteins from other species. Phylogenetic analysis showed that RpMYB4 had the closest relationship with FtMYB8 and was clustered in the S4 subfamily. Subcellular localization by confocal microscopy showed that an RpMYB4-GFP-fusion protein localized to the nucleus in tobacco. Real-time fluorescence quantitative PCR analyses revealed that RpMYB4 was differentially expressed in various tissues, with the highest expression in leaves, followed by petioles, rhizome, and roots, and with the lowest level in mature seeds. After treatment of R. palmatum L. seedlings with 200 μmol·L-1 MeJA, the expression of RpMYB4 in leaves was down-regulated within 24 h, and significantly up-regulated after 200 μmol·L-1 SA treatment at 12 h and 24 h. However, gene expression did not change with 200 μmol·L-1 ABA treatment. The transcripts of RpMYB4 under drought, high temperature, and mechanical injury stresses reached a peak at 24 h, 24 h, and at 3 h, respectively, while RpMYB4 expression was inhibited by low temperature stress, reaching its lowest value at 6 h. The gene showed no significant response to salt stress. Overall, RpMYB4 was cloned from R. palmatum L. for the first time, showed high expression in leaves, and was responsive to SA and various abiotic stress treatments including drought, high temperature, and mechanical injury. The results will be useful for further analysis of secondary metabolism and stress adaptations in R. palmatum L.
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Objective: To study the chemical constituents of tannin from the roots of Rheum palmatum. Methods: Chemical constituents of tannins were isolated by silica gel column chromatography, medium pressure column chromatography, and semi-preparative HPLC from the roots of R. palmatum. The structures were elucidated on the basis of spectral data analysis. Results: Four tannins, 3-O-cinnamoyl-1-O-galloyl-β-D-glucopyranoside (1), 2-O-cinnamoyl-1-O-galloyl-β-D-glucopyranoside (2), 2-O-cinnamoyl-1,6-di-O-galloyl-β-D-glucopyranoside (3), and 6-O-cinnamoyl-1-O-galloyl-β-D-glucopyranoside (4) were isolated from the roots of R. palmatum. Conclusion: Compound 1 is a new compound named palmatoside.
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Objective: To study the seeds shapes and germination characteristics, construct aseptic seedlings and callus cultural system, and provide a basis for the rapid propagation and secondary metabolic regulation of Rheum palmatum. Methods: Ten batches of R. palmatum seeds were subjected to characteristic analyses from the aspects of size, purification, weights per thousand seeds, seeds vigor, germination rates, and germination energy. The optimum disinfection system for aseptic seedlings and the optimum hormone ratio for inducing callus were screened by orthogonal test. The content of ten active components in aseptic seedlings and calli were primarily evaluated by HPLC analysis. Results: There was no significant difference in the appearances of the ten batches of R. palmatum seeds from different regions. The content of moisture, seeds vigor, germination rates, and germination energy differed apparently. The germination characteristics in Hezheng and Weiyuan counties were the best. The best disinfection group for aseptic seedlings was the combination of 75% ethanol for 30 s with 10% hydrogen peroxide for 15 min. The optimum hormones for callus induction were 6-BA (1.0 mg/L) + KT (2.0 mg/L) + NAA (1.5 mg/L). Seeds treated with different disinfectants had no significant effect on the content of ten components in germinated aseptic seedlings (P > 0.05). Seven active components were detected in callus, which was significantly lower than that in aseptic seedlings. And the content of chrysophanol-8-O-glucoside was the highest in the callus. Conclusion: The seed characteristics from Hezheng and Weiyuan counties in Gansu Province were excellent by analyses of weights per thousand seeds, seeds vigor, germination rates, and germination energy. The aseptic seedlings and callus cultural system of R. palmatum were successfully established, laying a solid foundation for further study.
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Anthraquinones are not only the main active constituents but also the index components for the quality control of Rhei Radix et Rhizoma. To study the anthraquinone biosynthesis, Rheum palmatum L. seedlings were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM 2000 150PE. The Illumina sequencing generated a total of 11.04 G clean data resulting in 736 309 74 clean reads, deposited in the sequence read archive (SRA accession SRP160030). Trinity do novo assembly yielded 93 646 unigenes, with an average of 1 108 nt. Functional annotation revealed that all unigenes were successfully annotated in the NR, NT, Swiss-port, PFAM, and KOG databases. GO enrichments showed that 57 subgroups were involved in biological process, cellular component, and molecular function. KEGG analysis indicated that 1 107 unigenes were implicated in 19 standard secondary metabolic pathways. 172 unigenes were analyzed to encode 28 key enzymes during the MVA, MEP, shikimic acid, and polyketide pathways related to anthraquinone biosynthesis. 125 CYP450 and 73 UGTs unigenes were related the modification of secondary metabolites in R. palmatum L. Furthermore, seven unigenes with full length cDNAs were successfully verified by RT-PCR and sequencing analyses. Then, MISA prediction produced a number of 18 885 simple sequence repeats (SSRs). Herein, the transcriptomic gene expression profiles of R. palmatum L. and candidate genes during the anthraquinone biosynthesis pathway were obtained for the first time. The results provided basic information for subsequent gene function characterization, secondary metabolic pathway analysis, and anthraquinone biosynthesis and regulation elucidation in R. palmatum L.
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Objective To compare the quality of Rheum palmatum L. in different areas and ages in Gansu Province by comparing five components in Rheum palmatum L. through fingerprints and QAMS (quantitative analysis of multi-components) methods; To determine the most suitable growing areas and the best development areas in Gansu Province. Methods The water content, total ashes, and water-soluble extract content in 15 batches of Rheum palmatum L. from different areas and ages in Gansu Province were determined according to the approaches listed in the Chinese Pharmacopeia. The contents of five index components were determined through HPLC and the quality of 15 batches of Rheum palmatum L were evaluated by fingerprint and QAMS method. Results The results from 15 batches of Rheum palmatum L. showed that the water content was less than 15％, total ashes less than 10％, and water-soluble extract content less than 25％, conforming to Pharmacopoeia. There were 23 common peaks in the fingerprints of 15 batches of Rheum palmatum L.. Five of them were identified as aloe-emodin, rhein, emodin, chrysophanol and physcion, which showed good linear relationship in the range of 0.0122–0.7344 μg (r=0.9999), 0.00714–5.7120 μg (r=0.9999), 0.0088–0.7040 μg (r=1.0000), 0.1224–3.6720 μg (r=0.9999) and 0.0148–5.9200 μg (r=0.9997), respectively. There was no significant difference between the calculated value and the measured value by using the relative correction factor (RCF), and the reproducibility of RCF was good. The quality of Rheum palmatum L. from different areas and ages in Gansu Province significantly differed (P<0.05). Two year old Rheum palmatum L. from Pingxiang village of Li county and Rushu village of Tanchang county had the best quality, and that of three years old was better than two years old from Tanchang county. Conclusion The established fingerprints and QAMS method is accurate, feasible, and can be used for the quality comparison of Rheum palmatum L. from different areas and ages in Gansu Province. Li County and Tanchang County areas in Gansu Province can be used as suitable planting areas and development industry. Therefore, it is recommended to select more than two yeas old Rheum palmatum L. from Tanchang County and Li County in Gansu Province for medicinal application.
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Objective: To analyze the main components in different parts of Rheum palmatum. Methods: The anthraquinones, soluble polysaccharides, cellulose, and mineral elements in the taproots, root heads, fibrous roots, root barks, petioles and leaves were detected by HPLC, UV, Weende, and ICP-AES. Results: The contents of aloe-emodin, rhein, emodin, chrysophanol, and physcion in taproots, root heads, fibrous roots, and root barks were 3.22-4.33, 1.33-2.32, 3.21-3.68, 3.22-3.76 mg/g, 0.77-1.36, 2.46-2.52, 1.16-1.46, 1.02-1.21 mg/g, 0.27-0.39, 0.28-0.34, 0.30-0.42, 0.31-0.67 mg/g, 2.85-3.70, 2.78-3.01, 4.02-4.81, 4.05-4.72 mg/g and 1.88-2.44, 1.82-2.01, 2.48-3.02, 3.61-4.46 mg/g, respectively. The contents of aloe-emodin, rhein, and emodin in leaves were 0.56-1.07, 0.45-0.69, 1.41-1.91 mg/g. The soluble polysaccharides in the taproots, petioles and leaves were 9.76%-10.42%, 5.76%-7.63%, and 3.50%-5.72%. The cellulose contents in petioles and leaves were 15.54% and 10.20%. Ca was the most abundant with 88.53 mg/g in leaves, followed by K with 32.42 mg/g, Mg with 12.93 mg/g, Al with 1.22 mg/g, and Fe with 1.17 mg/g. In the petioles, Ca with 80.60 mg/g and K with 28.73 mg/g were higher than those in roots with 21.08 and 14.09 mg/g. Na with 2.66 mg/g was also higher than that in roots with 0.26 mg/g and in leaves with 0.57 mg/g. Conclusion: The types and contents of anthraquinones in roots are higher than those in petioles and leaves, with the understanding of traditional medicinal parts. Emodin in leaves is five times as those in roots, petioles, and leaves, and also contains a certain amount of cellulose and soluble polysaccharide component, a wide variety of elements. From above analysis, the petioles and leaves could be deeper utilized.
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Objective To compare the effects of different extracts of Rheum palmatum on weight and tissue structure of hypothalamusand and pituitary of adult female rats,and screen the main reproductive toxicity extract.Methods The water,chloroform,ethyl acetate,and n-butanol extracts and water-soluble substance of R.palmatum were prepared by polarity gradient extraction method.Female adult rats were randomly divided into blank control group,rhubarb water extract group,and different extracts groups.The dosage of all the groups was equivalent to 4.00 g/kg crude rhubarb.Rats were administered with extracts by gavage for 60 d.Body mass growth rate of rats were calculated before and after administration.The pathological changes of hypothalamic arcuate nucleus neurons and pituitary gonadal cell were observed with light microscope.Results Compared with the blank control group,the body mass growth rate of rhubarb water extract group was decreased (P ＜ 0.05),while those in the different extracts groups were increased (P ＜ 0.01);The hypothalamic arcuate nucleus neurons of rhubarb water extract group showed chromatin marginalization,nissl substance dissolving,fuzzy boundary of nuclear membrane,as well as hell cells,and the total number of adenohypophysis cells reduced and the cells arranged in irregular.However,there were no apparent pathologic changes in different extracts groups.Conclusion Rhubarb water extract administration by long-term dose can reduce weight growth rate and result in pathologic changes of hypothalamic arcuate nucleus and adenohypophysis,while the different extracts can increase weight growth rate significantly and have little effects on the organizational structure ofhypothalamic arcuate nucleus and adenohypophysis.
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Hepatitis B virus(HBV) infection is a severe health problem in the world.However,there is still not a satisfactory therapeutic strategy for the HBV infection.To search for new anti-HBV agents with higher efficacy and less side-effects,the inhibitory activities of traditional Chinese medicine Rheum palmatum L.ethanol extract(RPE) against HBV replication were investigated in this study.Quantitative real-time polymerase chain reaction(PCR) was employed to analyze the inhibitory activity of RPE against HBV-DNA replication in a stable HBV-producing cell line HepAD38; the expression levels of HBV surface antigen(HBsAg) and e antigen(HBeAg) were also determined by enzyme linked immunosorbent assay(ELISA) after RPE treatment.RPE could dose-dependently inhibit the production of HBV-DNA and HBsAg.The concentration of 50% inhibition(IC50) was calculated at 209.63,252.53 μg/mL,respectively.However,its inhibitory activity against HBeAg expression was slight even at high concentrations.RPE had a weak cytotoxic effect on HepAD38 cells(CC50 = 1 640 μg/mL) and the selectivity index(SI) was calculated at 7.82.Compared with two anthraquinone derivatives emodin and rhein,RPE showed higher ability of anti-HBV and weaker cytotoxicity.So Rheum palmatum L.might possess other functional agents which could effectively inhibit HBV-DNA replication and HBsAg expression.Further purification of the active agents,identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.
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Object To study the effect of rare-earth elements La3+ on growth of hairy roots and normal roots of Rheum palmatum L and their yield of anthraquinones. Methods The biomass and an- thraquinone yield of two hairy clones DH5c, DH7a, and normal root NOR cultures of R. palmatum were evaluated statistically after various concentration of La3+ were administrated into culture medium. Results Significant differences of biomass yield and anthraquinone yield were shown among six La3+ concentrations, in which 10 mg/L showed obvious inhibitory effect for root growth and 1. 0 mg/L was the best for anthraquinone yield. In general, aloe-emodin and rhein were predominant in five anthraquinones after treated with the rare-earth element. Conclusion The growth of hairy root cultures of R. palmatum and their anthraquinone yield are greatly promoted by addition of La3+ to the medium.
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Objective To investigate the biotransformation of podophyllotoxin by cell suspension culture and root culture systems of Rheum palmatum.Methods Plant tissue culture technology was employed.The products were isolated by silica gel column chromatography and their structures were elucidated by spectral methods.Results Adding of podophyllotoxin had no obvious effects on the pH value of cell suspension culture and root culture systems of R.palmatum.In the case of present study,73.8% of podophyllotoxin was converted by cell suspension culture and 56.3% by root culture.The products were different between the two culture system.Podophyllotoxin was converted into picropodophyllin with cell suspension culture system and many other products besides epipodophyllotoxin with root culture system.Conclusion Both of cell suspension culture and root culture systems of R.palmatum are able to convert podophyllotoxin.
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Objective To study the effect of optimized media and clones on growth and anthraquinone yield in hairy root cultures of Rheum palmatum.Methods The four monoclones and four media were optimized statistically for studying the effect on the biomass accumulation and five anthraquinones yield in hairy root cultures of R.palmatum.Results Significant differences in biomass were shown among various clones and media,in which monoclone DH7a was the highest and WP medium was the best.Chrysophanol and physcion were predominant among five free anthraquinones in hairy root cultures of R.palmatum.Notable differences of total five free anthraquinones yield were observed among different clones, in which DH7a was the highest.Reciprocity analysis showed that the highest yield of five free anthraquinones is in the combinations of DH7a with B5 medium.DH5a less and DH5a the least with WP medium,DH7a and B5 medium were predominent for the yield of above five free anthraquinones with exception of DH5c which has no significant difference in four media.Conclusion The study lays the foundation for mass production of anthraquinone by hairy root culture of R.palmatum.