RESUMO
BACKGROUND: The apoptosis of pulmonary artery endothelial cells (PAECs) is an important factor contributing to the development of pulmonary hypertension (PH), a serious cardio-pulmonary vascular disorder. Salidroside (SAL) is a bioactive compound derived from an herb Rhodiola, but the potential protective effects of SAL on PAECs and the underlying mechanisms remain elusive. PURPOSE: The objective of this study was to determine the role of SAL in the hypoxia-induced apoptosis of PAECs and to dissect the underlying mechanisms. STUDY DESIGN: Male Sprague-Dawley (SD) rats were subjected to hypoxia (10% O2) for 4 weeks to establish a model of PH. Rats were intraperitoneally injected daily with SAL (2, 8, and 32 mg/kg/d) or vehicle. To define the molecular mechanisms of SAL in PAECs, an in vitro model of hypoxic cell injury was also generated by exposed PAECs to 1% O2 for 48 h. METHODS: Various techniques including hematoxylin and eosin (HE) staining, immunofluorescence, flow cytometry, CCK-8, Western blot, qPCR, molecular docking, and surface plasmon resonance (SPR) were used to determine the role of SAL in rats and in PAECs in vitro. RESULTS: Hypoxia stimulation increases AhR nuclear translocation and activates the NF-κB signaling pathway, as evidenced by upregulated expression of CYP1A1, CYP1B1, IL-1ß, and IL-6, resulting in oxidative stress and inflammatory response and ultimately apoptosis of PAECs. SAL inhibited the activation of AhR and NF-κB, while promoted the nuclear translocation of Nrf2 and increased the expression of its downstream antioxidant proteins HO-1 and NQO1 in PAECs, ameliorating the hypoxia-induced oxidative stress in PAECs. Furthermore, SAL lowered right ventricular systolic pressure, and decreased pulmonary vascular remodeling and right ventricular hypertrophy in hypoxia-exposed rats. CONCLUSIONS: SAL may attenuate the apoptosis of PAECs by suppressing NF-κB and activating Nrf2/HO-1 pathways, thereby delaying the progressive pathology of PH.
Assuntos
Apoptose , Células Endoteliais , Heme Oxigenase (Desciclizante) , Artéria Pulmonar , Transdução de Sinais , Animais , Masculino , Ratos , Apoptose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Glucosídeos/farmacologia , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/metabolismo , Rhodiola/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Two new guaiane sesquiterpenoids were isolated from the dried aerial parts of Dracocephalum tanguticum Maxim., named as dracotangusions A (1) and B (2), together with four known sesquiterpenoids, which were identified as Curcumenone (3), (4Z,7Z,9Z)-11-Hydroxy-4,7,9-germacratriene-1,6-dione (4), Kobusone (5), and (1S,10S), (4S, 5S)-(+)-germacrone-1(10)-4-diepoxide (6). The structures of isolates were determined by UV, IR, HR-ESI-MS, and NMR analysis. What is noteworthy is that four known sesquiterpenoids were isolated for the first time from the genus of Dracocephalum L. All compounds inhibited the extremely significant difference (p < 0.01) in anti-inflammatory activity, suggesting that these compounds may be promising candidates as an anti-inflammatory agent.
RESUMO
One galactose- and arabinose-rich polysaccharide isolated from Sambucus adnata was named SPS-1, which had an average molecular weight 138.52 kDa, and was composed of L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-galactose, and L-arabinose in a molar ratio of 0.6:0.4:0.1:4.9:4.0. The primary structure of SPS-1 was further analyzed through methylation and NMR spectroscopy. The results showed that SPS-1 had the structural characteristics of AG-II pectin. The immunoactivity test showed that SPS-1 activated the phosphorylation of MAPKs-related proteins and further elevated the expression levels of related nuclear transcription factors (IκBα and NF-κB p65) in the cells through the TLR2 and MyD88/TRAF6-dependent pathway, thereby significantly enhancing the phagocytosis of macrophages and stimulating the secretion of NO, IL-1ß, IL-6, and TNF-α, which activated the RAW264.7 cells. Therefore, SPS-1, acting as an immunomodulator, is a potential drug for immunological diseases.
Assuntos
Sambucus , Animais , Arabinose , Galactose , Camundongos , Polissacarídeos/química , Células RAW 264.7RESUMO
Three undescribed prenylated benzoic acid derivatives berberisides A-C (1-3) and a new clerodane glycoside berberiside D (4) were isolated from Berberis tsarica Aherndt. Their structures were elucidated on the basis of extensive NMR and HR-ESI-MS analysis. The in vitro cytotoxic activities of all isolates were studied against lung carcinoma A549, hepatocellular carcinoma HepG2 and breast carcinoma MDA-MB-231 cell lines. Among them, compounds 1 and 4 exhibited anti-proliferative effects against three tumor cell lines with IC50 ranging from 28.97 ± 2.18 to 35.83 ± 0.72 µM.
Assuntos
Berberis , Diterpenos Clerodânicos , Ácido Benzoico , Linhagem Celular Tumoral , Diterpenos Clerodânicos/farmacologia , Glicosídeos/farmacologia , Humanos , Estrutura MolecularRESUMO
Paeonia ludlowii, a plant of the Paeoniaceae family, has abundant genetic diversity in different populations, and the seed oil can be used in a diverse number of activities. However, its neuroprotective effect is not clear. We investigated the memory-improving effects and associated mechanisms of Paeonia ludlowii seed oil (PLSO) on amyloid beta (Aß)25-35-induced Alzheimer's disease (AD) in rats. The Morris water maze test was undertaken, and subsequently, the content of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and acetylcholinesterase (ACHE) in the hippocampus was detected by biochemical analyses. To further study PLSO, we examined the pathologic structure and apoptosis of hippocampal tissue by staining. Immunohistochemical analysis was used to detect expression of IBA-1 and GFAP in the hippocampus. Detection of proinflammatory factors was achieved by reverse transcription-quantitative polymerase chain reaction and Western blotting. High-dose PLSO inhibited expression of GFAP and IBA-1. We demonstrated that high-dose PLSO can regulate activation of glial cells and mediate apoptosis of hippocampal cells, and significantly improve learning and memory deficits in AD rats. PLSO could be developed as a nutritional supplement and sold as a drug for AD prevention and/or treatment.
RESUMO
This paper aims to investigate the chemical constituents of the seeds of Herpetospermum pedunculosum. One new coumarin and two known lignans were isolated from the ethanolic extract of the seeds of H. pedunculosum with thin layer chromatography(TLC), silica gel column chromatography, Sephedax LH-20 chromatography, Semi-preparative high performance liquid chromatography and recrystallization, etc. Their structures were elucidated as herpetolide H(1), phyllanglaucin B(2), and buddlenol E(3) by analysis of their physicochemical properties and spectral data. Among them, compound 1 was a new compound, and compounds 2 and 3 were isolated from this genus for the first time. In vitro anti-inflammatory activity test showed that herpetolide H had certain NO inhibitory activity for LPS-induced RAW 264.7 cells, with its IC_(50) value of(46.57±3.28) µmol·L~(-1).
Assuntos
Cucurbitaceae , Lignanas , Cromatografia Líquida de Alta Pressão , Cumarínicos/farmacologia , SementesRESUMO
Two undescribed sesamin-type sesquilignans ptehoosines A (1) and B (2), together with 4 known lignans (3-6), were isolated from Pterocephalus hookeri (C.B. Clarke) Höeck which was widely used as traditional Tibetan medicine for treatment of rheumatoid arthritis. Their structures were determined by HR-ESI-MS, NMR analysis and CD experiment. The in vitro antiangiogenic effect of all isolated compounds against human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8 assay. Among them, compound 1 exhibited significant proliferative inhibition on HUVECs with IC50 value of 32.82 ± 0.99 µM. Further in vitro study indicated 1 could arrest cell cycle at G0/G1 phase and reduce the migration of HUVECs. In vivo experiment exhibited 1 could inhibit tail vessels plexus in zebrafish. The above finding suggested that 1 was a promising lead compound against RA by inhibiting of angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Caprifoliaceae/química , Dioxóis/farmacologia , Lignanas/farmacologia , Inibidores da Angiogênese/isolamento & purificação , Animais , Pontos de Checagem do Ciclo Celular , Dioxóis/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Lignanas/isolamento & purificação , Medicina Tradicional Tibetana , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Tibet , Peixe-ZebraRESUMO
To determine the food potential of Paeonia ludlowii D.Y.Hong (P. ludlowii) kernel oil, in this study, we analysed the fatty acid composition and volatile components of this oil, compared the antioxidant effects of two natural antioxidants on it, and then predicted its shelf life at room temperature (25°C). The results showed that P. ludlowii kernel oil mainly contained 20 fatty acids, of which linoleic acid, oleic acid and other unsaturated fatty acid contents together made up 86.99%. The aromatic composition of the crude P. ludlowii kernel oil was analysed, and 34 aromatic compounds were obtained, including 5 lipids (2.30%), 9 alcohols (12.64%), 6 aldehydes (14.67%), 2 alkanes (1.30%), 5 acids (2.70%), 1 ketone (0.41), 2 alkenes (39.12%) and 4 other substances (26.85%). The effects of the antioxidants were ranked as follows: 0.04% tea polyphenols + crude oil > 0.04% bamboo flavonoids + crude oil > crude oil. In addition, the shelf lives at room temperature (25â) of each kernel oil-antioxidant mixture were 200.73 d, 134.90 d and 131.61 d, respectively. Overall, these results reveal that P. ludlowii kernel oil is a potential candidate for a new high-grade edible oil, and its development has broad application prospects.
Assuntos
Ácidos Graxos Insaturados/análise , Ácidos Graxos Voláteis/análise , Qualidade dos Alimentos , Armazenamento de Alimentos , Paeonia/química , Óleos de Plantas/química , Antioxidantes , Ácidos Graxos Insaturados/química , Ácidos Graxos Voláteis/química , Ácido Linoleico/análise , Ácido Oleico/análise , Óleos de Plantas/isolamento & purificação , Temperatura , Fatores de TempoRESUMO
By the fourth survey of Chinese medicinal resources, new medicinal plants records of 2 genera and 5 species were reported in Tibet. They are two genera Rhynchoglossum and Asteropyrum, and five species including Rh. obliquum, A. peltatum, Urena repanda, Schefflera khasiana and Mimulus tenellus. All the voucher specimens are preserved in Herbarium of Tibet Agriculture and Animal Husbandry University.
Assuntos
Araliaceae/classificação , Lamiales/classificação , Malvaceae/classificação , Plantas Medicinais/classificação , Ranunculaceae/classificação , TibetRESUMO
Artemisinin is a preferred medicine in the treatment of malaria. In this study, AaCMK, a key gene involved in the upstream pathway of artemisinin biosynthesis, was cloned and characterized from Artemisia annua for the first time. The full-length cDNA of AaCMK was 1 462 bp and contained an ORF of 1 197 bp that encoded a 399-anomo-acid polypeptide. Tissue expression pattern analysis showed that AaCMK was expressed in leaves, flowers, roots and stems, but with higher expression level in glandular secretory trichomes. In addition, the expression of AaCMK was markedly increased after MeJA treatment. Subcellular localization showed that the protein encoded by AaCMK was localized in chloroplast. Overexpression of AaCMK in Arabidopsis increased the contents of chlorophyll a, chlorophyll b and carotenoids. These results suggest that AaCMK plays an important role in the biosynthesis of terpenoids in A. annua and this research provids a candidate gene that could be used for engineering the artemisinin biosynthesis.
Assuntos
Artemisia annua/genética , Proteínas de Plantas/genética , Artemisia annua/enzimologia , Artemisininas , Clorofila A , Clonagem MolecularRESUMO
The aerial parts of Epilobium plants are widely used as folk medicine and food around the world. The present study was aimed to investigate the antioxidant activities and active chemical constituents from Epilobium angustifolium L. The results revealed that the EtOAc extract, rich in phenolic compounds and flavonoids (16.81 ± 0.67 g GAE/100 g extract and 4.95 ± 0.21 g QE/100 g extract, respectively), possessed significantly antioxidant activities in reducing power, DPPH radical scavenging activity, ABTS radical scavenging activity and highly in inhibiting lipid peroxidation activity. Simultaneously, active fractions F to H from EtOAc extracts showing potent in vitro antioxidant activities also contained high content of total phenolic and flavonoid. Twenty-eight compounds were identified as phenolic compounds and flavonoids by LC-MS/MS. The results illustrate that the E. angustifolium L., which is rich in phenolics, could be used as a natural resource of antioxidant ingredient.
Assuntos
Antioxidantes/farmacologia , Epilobium/química , Fenóis/análise , Extratos Vegetais/farmacologia , Antioxidantes/química , Cromatografia Líquida de Alta Pressão/métodos , Flavonoides/análise , Flavonoides/química , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fenóis/química , Componentes Aéreos da Planta/química , Extratos Vegetais/análise , Extratos Vegetais/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Mirabilis himalaica (Edgew.) Heimerl is among the most important genuine medicinal plants in Tibet. However, the biosynthesis mechanisms of the active compounds in this species are unclear, severely limiting its application. To clarify the molecular biosynthesis mechanism of the key representative active compounds, specifically rotenoid, which is of special medicinal value for M. himalaica, RNA sequencing and TOF-MS technologies were used to construct transcriptomic and metabolomic libraries from the roots, stems, and leaves of M. himalaica plants collected from their natural habitat. As a result, each of the transcriptomic libraries from the different tissues was sequenced, generating more than 10 Gb of clean data ultimately assembled into 147,142 unigenes. In the three tissues, metabolomic analysis identified 522 candidate compounds, of which 170 metabolites involved in 114 metabolic pathways were mapped to the KEGG. Of these genes, 61 encoding enzymes were identified to function at key steps of the pathways related to rotenoid biosynthesis, where 14 intermediate metabolites were also located. An integrated analysis of metabolic and transcriptomic data revealed that most of the intermediate metabolites and enzymes related to rotenoid biosynthesis were synthesized in the roots, stems and leaves of M. himalaica, which suggested that the use of non-medicinal tissues to extract compounds was feasible. In addition, the CHS and CHI genes were found to play important roles in rotenoid biosynthesis, especially, since CHS might be an important rate-limiting enzyme. This study provides a hypothetical basis for the screening of new active metabolites and the metabolic engineering of rotenoid in M. himalaica.
Assuntos
Perfilação da Expressão Gênica/métodos , Metabolômica/métodos , Mirabilis/genética , Mirabilis/metabolismo , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Espectrometria de Massas , Redes e Vias Metabólicas , Anotação de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Análise de Sequência de RNARESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The ripe seeds of Herpetospermum caudigerum have been used in Tibetan folk medicine for treatment of bile or liver diseases including jaundice, hepatitis, intumescences or inflammation. Previously reports suggested that the seed oil and some lignans from H. caudigerum exhibited protective effects against carbon tetrachloride (CCl4)-induced hepatic damage in rats, which may be related to their free radical scavenging effect. However, the protective effect of H. caudigerum against cholestasis is still not revealed. The aim of the present study was to investigate the pharmacological effect and the chemical constituents of the petroleum ether extract (PEE) derived from H. caudigerum against α-naphthylisothiocyanate (ANIT)-induced acute cholestasis in rats. MATERIALS AND METHODS: Male cholestatic Sprague-Dawley (SD) rats induced by ANIT (60mg/kg) were orally administered with PEE (350, 700 and 1400mg/kg). Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyl transpeptidase (γ-GTP), total bilirubin (TBIL), direct bilirubin (DBIL) and total bile acid (TBA), as well as bile flow, and histopathological assay were evaluated. Hepatic malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione S-transferase (GST), and nitric monoxide (NO) in liver were measured to explore the possible protective mechanisms. Phytochemical analysis of PEE was performed by gas chromatography-mass spectrometer (GC-MS). RESULTS: PEE have exhibited significant and dose-dependent protective effect on ANIT-induced liver injury by reduce the increases in serum levels of ALT, AST, ALP, γ-GTP, TBIL, DBIL and TBA, restore the bile flow in cholestatic rats, and reduce the severity of the pathological tissue damage induced by ANIT. Hepatic MDA, MPO and NO contents in liver tissue were reduced, while SOD and GST activities were elevated in liver tissue. 49 compounds were detected and 39 of them were identified by GC-MS analysis, in which long-chain fatty acids were the main constituents. CONCLUSIONS: PEE exhibited a dose-dependently protective effect on ANIT-induced liver injury in cholestatic rats with the potential mechanism of attenuated oxidative stress in the liver tissue, and the possible active compounds were long-chain fatty acids.
Assuntos
1-Naftilisotiocianato/toxicidade , Colestase/tratamento farmacológico , Cucurbitaceae , Medicina Tradicional Tibetana , Extratos Vegetais/uso terapêutico , Doença Aguda , Animais , Colestase/induzido quimicamente , Colestase/metabolismo , Cucurbitaceae/química , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/análise , Ratos , Ratos Sprague-DawleyRESUMO
One new compound, himalain A (1), together with 12 known compounds were isolated from Mirabilis himalaica. Their structures were determined by spectroscopic methods, including 2D NMR techniques, and the absolute configuration of the 1,2-diol moiety in 1 was defined through Riguera's method. All isolated compounds were tested for their cytotoxic and antibacterial activities.
Assuntos
Mirabilis/química , Éteres Fenílicos/isolamento & purificação , Antibacterianos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Humanos , Éteres Fenílicos/química , Éteres Fenílicos/farmacologiaRESUMO
Tropane alkaloids are anticholinergic drugs widely used clinically. Biosynthesis of tropane alkaloids in planta involves a step of transamination of phenylalanine. Based on the sequenced transcriptomes of lateral roots and leaves of Hyoscyamus niger, we found three annotated aromatic amino acid aminotransferases, which were respectively named HnArAT1, HnArAT2 and HnArAT3. Sequence analysis showed that HnArAT3 had highest similarity with the reported Atropa belladonna Ab Ar AT4, which was involved in tropane alkaloid(TA) to provide the precursor of the phenyllactic acid moiety. Tissue expression pattern analysis indicated that HnArAT3 was specifically expressed in lateral roots, where is the organ synthesizing tropane alkaloids. Then, method of virus induced gene silencing (VIGS) was used to characterize the function of HnArAT3 in H. niger. Gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had lower expression levels of HnArAT3 than the non-transgenic control, and HPLC analysis of alkaloids demonstrated significant decrease in the contents of hyoscyamine, anisodamine and scopolamine in planta. These results suggested that HnArAT3 was involved in the phenyllactic acid branch of TA biosynthetic pathway. Molecular cloning and functional identification of HnArAT3 laid the foundation for further understanding of TA biosynthesis and metabolic regulation, and also provided a new candidate gene for engineering biosynthetic pathway of tropane alkaloids.
Assuntos
Alcaloides/biossíntese , Hyoscyamus/genética , Proteínas de Plantas/genética , Transaminases/genética , Tropanos/metabolismo , Atropa belladonna , Vias Biossintéticas , Antagonistas Colinérgicos , Clonagem Molecular , Hiosciamina , Hyoscyamus/enzimologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Escopolamina , Alcaloides de SolanáceasRESUMO
One new cyanoside, rhobupcyanoside B (1), together with 7 known ones, was isolated from the 70% ethanol extract of the roots and rhizomes of Rhodiola bupleuroides. Their structures were determined by spectroscopic methods, including 2D NMR techniques. Compound 1 was evaluated for its inhibitory activity against α-glucosidase with IC50 value of 278.28 ± 0.55 µM by comparing with the positive control (acarbose) at 210.40 ± 0.32 µM.
Assuntos
Glicosídeos/isolamento & purificação , Rhodiola/química , alfa-Glucosidases/efeitos dos fármacos , Inibidores de Glicosídeo Hidrolases/química , Glicosídeos/química , Glicosídeos/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Raízes de Plantas/químicaRESUMO
The chemical constituents of Herpetospermum caudigerum were investigated using chromatographic methods, including silica gel column chromatography, Sephadex LH-20 and semi-preparative HPLC. Four compounds were isolated and their structures were elucidated by spectral data and physicochemical properties, which were identified as 2,11-dimethoxy-3,9-dihydroxy-7 H-dibenzo[c,e]oxepin-5-one (1),7,8'-didehydroherpetotriol(2), herpetotrio l(3) and kaempferitrin (4). Among those, compound 1 is one new 7H-dibenzo[c,e]oxepin-5-one, named as herpetolide C.
Assuntos
Cucurbitaceae/química , Oxepinas/isolamento & purificação , Cromatografia Líquida de Alta PressãoRESUMO
A new isocoumarin, chamerilactone A(1) was isolated from the ethanol extract of Chamerion angustifolium with normal phase silica column chromatography, Sephadex LH-20, MCI CHP-20 and semi- preparative HPLC methods. Its structure and stereochemistry were elucidated by spectroscopic methods, including 2D-NMR and optical rotation techniques.
Assuntos
Isocumarinas/isolamento & purificação , Onagraceae/química , Cromatografia Líquida de Alta Pressão , Estrutura MolecularRESUMO
The plastidial methylerythritol phosphate(MEP) pathway provides 5-carbon precursors to the biosynthesis of isoprenoid (including artemisinin). 2-C-Methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT) is the third enzyme of the MEP pathway, which catalyzes 2-C-methyl-D-erythritol-4-phosphate to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol. The full-length MCT cDNA sequence (AaMCT) was cloned and characterized for the first time from Artemisia annua L. Analysis of tissue expression pattern revealed that AaMCT was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. AaMCT was found to be a methyl jasmonate (Me JA)-induced genes, the expression of AaMCT was significantly increased after MeJA treatment. Subcellular localization indicated that the GFP protein fused with AaMCT was targeted specifically in chloroplasts. The transgenic plants of Arabidopsis thaliana with AaMCT overexpression exhibited a significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids, demonstrating that AaMCT kinase plays an influential role in isoprenoid biosynthesis.
Assuntos
Artemisia annua/enzimologia , Nucleotidiltransferases/genética , Proteínas de Plantas/genética , Acetatos , Arabidopsis , Artemisia annua/genética , Artemisininas , Carotenoides/análise , Clorofila/análise , Clorofila A , Clonagem Molecular , Ciclopentanos , DNA Complementar , Regulação da Expressão Gênica de Plantas , Oxilipinas , Plantas Geneticamente ModificadasRESUMO
Artemisinin is the first choice for malaria treatment. The plastidial MEP pathway provides 5-carbon precursors (IPP and its isomer DMAPP) for the biosynthesis of isoprenoid (including artemisinin). Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) is the last enzyme involved in the MEP pathway, which catalyzes HMBPP to form IPP and DMAPP. In this study, we isolated the full-length cDNA of HDR from Artemisia annua L. (AaHDR2) and performed functional analysis. According to gene expression analysis of AaHDR2 (GenBank: KX058541) and AaHDR1 reported ever (GenBank: ADC84348.1) by qPCR, we found that AaHDR1 and AaHDR2 had much higher expression level in trichomes than that in roots, stems, leaves and flowers. AaHDR2 had much higher expression level in flowers than that in leaves. Further, the plant hormones such as Me JA and ABA respectively up-regulated the expression level of AaHDR1 and AaHDR2 significantly, but GA3 up-regulated the expression level of AaHDR2 only. The gene expression analysis of AaHDR1 and AaHDR2 showed that AaHDR2 had a greater contribution than AaHDR1 to isoprenoid biosynthesis(including artemisinin). We used AaHDR2 for the following experiments. Bioinformatic analysis indicated that AaHDR2 belonged to the HDR family and the functional complementation assay showed that AaHDR2 did have the enzymatic function of HDR, using E. coli mutant MG1655(ara)<>HDR as host cell. The subcellular localization assay showed that AaHDR2 fused with GFP at its N-terminal specifically targeted in chloroplasts. Finally, AaHDR2 was overexpressed in Arabidopsis thaliana. The AaHDR2-overexpressing plants produced the isoprenoids including chlorophyll a, chlorophyll b and carotenoids at significantly higher levels than the wild-type Arabidopsis plants. In summary, AaHDR2 might be a candidate gene for genetic improvement of the isoprenoid biosynthesis.