ABSTRACT
Objective: To study the chemical constituents from the barks of Betula platyphylla. Methods: The chemical constituents were isolated by silica gel column chromatography and HPLC, and its structure were identified by their spectral data and physicochemical properties analysis. Results: A total of 20 compounds were isolated from B. platyphylla, which were identified as 3-oxo-24-methylenecycloartan (1), sitosterone (2), 3β-acetyl oleanolic acid (3), lupeol (4), citrostadienol (5), betulinaldehyde (6), 3β-acetoxyl-11α,12α-epoxy-oleanan-13β,28-olide (7), oleanolic acid (8), erythrodiol (9), 3-hydroxy-1,7-bis(4',4″- dihydroxyphenyl)- heptane (10), β-sitosterol (11), 3β,20-dihydroxylupane (12), 3β-O-caffeoyl-betulin (13), platanic acid (14), 3β,12α-dihydroxyoleanan- 13β,28-olide (15), 30-norlupane-3β,28-diol-20-one (16), betulinic acid (17), betulin (18), 1,7-bis (4',4″-dihydroxyphenyl)-hept- 4-ene-3-one (19), and 5-hydroxy-1,7-bis (4',4″-dihydroxyphenyl)-hept-3-one (20). Conclusion: Compounds 1, 5, 7, 12, 14-16 are isolated from B. platyphylla for the first time, and compounds 1, 5, 7, 14-16 are isolated from betula genus for the first time.
ABSTRACT
Objective: To analyze the effect of carbon monoxide (CO) on the growth of Betula platyphylla suspension cells and triterpenoid production. Methods: CO donor hematin of 5, 10, 20, and 30 μmol/L were added to eight-day-old B. platyphylla suspension cells, and the change of triterpenoid content and gene expression of FPPS, LUS, CAS1, CAS2, and β-AS related to the triterpenoid synthesis were analyzed by chemical colorimetry, high performance liquid chromatography and real-time PCR, respectively. Results: CO treatment did not have a significant effect on dry weight of B. platyphylla suspension cells except for 2-8 d at 20 μmol/L and 30 μmol/L CO treatment. CO treatment also did not have a significant effect on pH value and malondialdehyde content except for 30 μmol/L CO treatment. CO treatment significantly increased the content of triterpenoid, betulin and oleanolic acid. the content of triterpenoid, betulin and oleanolic acid were the highest after 1 d at 30 μmol/L CO treatment, 4 d at 10 μmol/L CO treatment and 2 d at 10 μmol/L CO treatment, which was 1.3, 1.5, and 4.2 times of the control, respectively. Triterpenoid production in B. platyphylla suspension cells induced by CO was further confirmed by key enzyme genes of FPPS, LUS, CAS1, CAS2, and β-AS related to the triterpenoid synthesis by RT-PCR. Conclusion: CO treatment could effectively enhance the triterpenoid production in B. platyphylla suspension cell culture.
ABSTRACT
Objective To study the full-length, promoter sequences and its coding structure and properties of thioredoxin (Trx) gene of Betula platyphylla (BpTRX), and reveal the expression pattern of BpTRX under H2S treatment. Methods The BpTRX gene was cloned by PCR, and the promoter region sequences of BpTRX was obtained by using chromosome walking technique. The BpTRX gene, the promoter and its encoded protein were analyzed by bioinformatics software. The phylogenetic tree of BpTRX was constructed. The expression patterns of BpTRX gene under H2S exogenous stress were analyzed by quantitative real-time PCR. Results The full-length of BpTRX gene is 351 bp, encoding 117 amino acids. The BpTRX gene was closely related to the TRX-H protein of castor bean, soybean, alfalfa and grape. The obtained BpTRX promoter region sequences are 873 bp, which contains the essential elements of transcription and a large number of stress response and hormone response elements. Conclusion The full length and partial promoter sequences of the BpTRX gene were obtained. The response trend of BpTRX gene to H2S treatment was in the form of bimodal, which was first rises, then decreases, then rises and then declines.
ABSTRACT
Objective: To study the full-length gene structure characteristics and coding protein properties of 3-hydroxy-3-methyl-glutaryl-CoA reductase (BpHMGR) from Betula platyphylla (birch). This reveals the mechanism of rhythm expression pattern and its signaling molecule in response to exogenous NO. Methods: The BpHMGR gene in birch was cloned by RACE technology. BpHMGR and its encoded protein were analyzed in detail by bioinformatics software. And the expression pattern in the circadian variation of BpHMGR gene was analyzed by real-time PCR. Results: The full-length of BpHMGR gene was 1764 bp, contained the complete ORF, encoding 587 amino acids (Genebank ID: KJ197336). According to BpHMGR amino acid sequence, phylogenetic tree was constructed. And the effect of circadian rhythm and exogenous NO on the transcription level of BpHMGR in the leaves of B. platyphylla was investigated. Conclusion: BpHMGR gene, soybean (XP_003519474.1), grape (XP_002275827.1), and alfalfa (XP_003617066.1) have close genetic relationship. BpHMGR changes with the diurnal cycle showing the expression characteristics of rhythm, and reaches the peak at 9 pm. The up-regulation expression of key enzyme gene HMGR in the exogenous NO-induced birch intracellular triterpenoids metabolic pathway is initially revealed, so as to promote the synthesis of triterpene oleanolic acid product.
ABSTRACT
Objective: Nitric oxide synthase (NOS) is a kind of important NOS in organisms. The gene cloning and expression pattern analysis of NOS in plant laid the foundation for study on the biological functions of nitric oxide in plants. Methods: NOS gene in B. platyphylla was cloned by RACE technology, named BpNOS. Bioinformatics and molecular evolution analysis, abiotic stress, and signal induced of the gene were preliminarily carried out. Results: This full-length gene was 1 806 bp with the complete ORF, encoding 601 amino acids (Genebank ID: KJ197336). BpNOS was an unstable hydrophobic protein. This protein may exist signal peptide with transmembrane ability. The alpha helix, extension chain, random coil distributed throughout the protein. Molecular evolution analysis results showed that the genetic distance of BpNOS gene was closer to the gene genetic distance of grape species NOS, which indicates the relatively close relationship between them; While the genetic distance of BpNOS gene was farther from soybean and alfalfa, which explains that the relationship of them is far. The expression of BpNOS has rhythm, reached the maximum at 15: 00, 12 times as at 0: 00. The expression of BpNOS gene could be inhibited by salt, low temperature, and heavy metal cadmium stress, but in a different response pattern. The expression of BpNOS gene could be promoted by salicylic acid and exogenous NO. Conclusion: Abiotic stress could inhibit the activity of NOS. The salicylic acid and exogenous NO could be synthesized by NOS pathway to regulate endogenous NO. This paper lays a solid foundation for the further research on the metabolic regulation of NO in B. platyphylla.
ABSTRACT
Objective: To analyze the effect of exogenous putrescine on triterpenoid accumulation and sucrose metabolism. Methods: Putrescines (0.1, 1, and 5 mmol/L) were added to the eight-day-old suspension cell culture, and the content changes of sucrose, fructose, glucose, and triterpenoid, the activity of sucrose phosphate synthase (SPS) and sucrose synthase (SS) were analyzed by colorimetry. Results: Putrescine increased the content of sucrose in cells but decreased the conent in culture medium. Among them, the content of sucrose was most enhanced after putrescine induction for 6 h; The increasing rate was 169.41%. However, the contents of fructose and glucose were only increased in 6 h under the putrescine treatment, the other treatments decreased the contents of fructose and glucose. In addition to 5 mmol/L putrescine treatment, putrescine enhanced the activities of SPS and SS. The content of triterpenoid in suspension cells was increased in 12 h after 5-40 g/L sucrose induction, and the content of triterpenoid was most induced by 20 g/L sucrose, the increasing rate was 52.16%. In addition to increasing 27.37% on triterpenoid content in cells treated by 1 mmol/L putrescine and 5 g/L sucrose, the treatment of 1 mmol/L putrescine and other sucrose concentration decreases the accumulation of triterpenoid. Conclusion: Putrescine could increase the sucrose content and triterpenoid accumulation in suspension cell culture, and we preliminarily inferred that the induction of sucrose by putrescine could participate in the accumulation of triterpenoid in birch cells.
ABSTRACT
Objective: To clarify whether polyamine-mediated triterpenoid synthesis in birch (Betula platyphylla) suspension cells induced by fungal elicitor. Methods: Fungal elicitor (40 μg/mL), putrescine (Put, 1 mmol/L), and D-arginine (D-Arg, 2 mmol/L) were added to the eight-day-old suspension cell culture, the content changes of triterpenoid and free polyamines were analyzed by chemical colorimetry and HPLC. The effect of polyamine on triterpenoid synthesis in B. platyphylla suspension cells induced by fungal elicitor was analyzed by pharmacology and restoration experiment. Results: After the treatment of fungal elicitor or Put, the contents and yields of free polyamines and triterpenoid were increased. Among of them, the triterpenoid content was the highest after 24 h treatment, the increasing rates were 68.54% and 30.34%, respectively. The triterpenoid content was increased by the cotreatment of fungal elicitor and Put, but the increasing degree of yield was lower than that by the treatment of fungal elicitor alone. Compared with the fungal elicitor alone, the cotreatment of fungal elicitor and D-Arg decreased the triterpenoid content by 40.57% in the highest degree of decreasing after 24 h treatment. In restoration experiment, with the treatment time prolonging, the effect of fungal elicitor, Put, or cotreatment of fungal elicitor and D-Arg on triterpenoid synthesis was decreasing to the control level finally. Conclusion: Polyamine could mediate the triterpenoid synthesis in cells of B. platyphylla induced by fungal elicitor.
ABSTRACT
Objective: To analyze the effect of exogenous polyamine on the growth of Betula platyphylla suspension cells and triterpenoid accumulation. Methods: At the end of the growth stage, 0.1 mmol/L and 1.0 mmol/L putrescine (Put), spermine (Spm), and spermidine (Spd) were added to the suspension cells of B. platyphylla, the triterpenoid content and gene expression of lupeol synthase (LUS) related to the synthesis of triterpenoid were analyzed by chemical colorimetry and RT-PCR, respectively. Results: Except for the treatment with 1.0 mmol/L Spm, the cell viability, dry weight, and triterpenoid production in B. platyphylla suspension cells induced by Put and Spd were increased, and the dry weight and triterpenoid production of B. platyphylla cells were increasing with the extension of polyamine treatment time. Among them, dry weight and triterpenoid production were the highest after 2 d Put induction, which were 38.89% and 116.35% higher than those in the control, respectively. Triterpenoid production in B. platyphylla cells induced by polyamine was further confirmed by RT-PCR of LUS gene. Conclusion: Put could effectively enhance the growth of B. platyphylla suspension cells and triterpenoid accumulation.
ABSTRACT
Objective: To analyze the interaction between putrescine (Put) and hydrogen peroxide (H2O2) on the regulation of triterpenoid production in Betula platyphylla suspension cell culture. Methods: Put (1 mmol/L), H2O2 (0.1 mmol/L), and fungal elicitors (40 μg/mL) were added into the eight-day-old B. platyphylla suspension cell culture, and the changes of triterpenoid content, polyamine content, and H2O2 fluorescence intensity were analyzed by chemical colorimetry, HPLC, and fluorescence microscopy, respectively. Results: Put (1 mmol/L) increased the H2O2 fluorescence intensity and triterpenoid content in B. platyphylla suspension cell culture. H2O2 (0.1 mmol/L) promoted the triterpenoid production, inhibited the production of spermidine (Spd) and spermine (Spm), and had no effectt on Put content. Under the treatment of 1 mmol/L Put and 0.1 mmol/L H2O2, H2O2 fluorescence intensity and polyamine (PAs) content were between its separated treatment, and triterpenoid content was significantly higher than its separated treatment. Comparing with the fungal elicitor, fungal elicitor and H2O2 scavenger catalase (CAT) induced an increase of 0.62% and 16.05% in Put and Spd content, respectively, while no effect on Spm content. Fungal elicitor and PAs synthesis inhibitor D-arginine (D-arg) decreased the H2O2 fluorescence intensity. Under the treatment of fungal elicitor, the fluorescence intensity of CAT and D-arg, H2O2 and the contents of PAs and triterpenoid were lower than those of fungal elicitor and CAT or D-arg, but triterpenoid content was still higher than that of the control. Conclusion: This can be inferred that the interaction between Put and H2O2 regulates the triterpenoid production in birch suspension cell culture.
ABSTRACT
Objective: Using three-year-old birch (Betula platyphylla) as the experimental material to study the changes of the accumulation of triterpenoid precursor and triterpenoid components in birch, and to analyze the fluctuation characteristics of triterpenoid component accumulation. Methods: Eight compositions, such as the total triterpene, betulin, flavonoids, etc in twig barks and leaves, were analyzed by HPLC and UV spectrophotometer. Results: The accumulative characteristics of each component in triterpenoid substances was significantly different between the twig barks and leaves. During the growing seasons (May-September), the accumulation of 2, 3-oxidized squalene, betulinic acid, betulin, oleanolic acid, dammarenediol, sterol, and total triterpene reached the peak in July-September. Among these, 2, 3-oxidized squalene had a lower content and its highest content was 0.067 mg/g in July. The content of betulinic acid was 8.34 mg/g, while the content of oleanolic acid reached 37.51 mg/g in September. The highest content of dammarenediol was 14.18 mg/g and the total triterpene content was 168.17 mg/g in July. The higher concentration of total triterpene in the leaves was 126.11 mg/g in August, while the contents of 2, 3-oxidized squalene, betulin, and oleanolic acid were very low and not detected. The accumulated peaks of flavonoids in twig barks and leaves were 53.02 and 58.49 mg/g, respectively in May and August. The fluctuation analysis showed that the accumulation of triterpenoid components did not coincide with that of total triterpene in twig barks and leaves,. The total triterpene, dammarenediol, oleanolic acid, and betulinic acid showed the S-shaped curve in twig barks, and the accumulation of betulin showed the inverted "V" curve, the while trend of flavonoid accumulation was contrary to the total triterpene. The trend of the total triterpene accumulation was similar to that of flavonoids, while the trends of dammarenediol and 2, 3-oxidized squalene accumulation were similar. Conclusion: This research identifies the accumulation and distribution characteristics of triterpenoid and flavonoids in the twig barks and leaves of three-year-old birch, and lays the foundation for triterpenoid resources discovering, sustainable use, and metabolic regulation in twig barks and leaves of birch.