RESUMO
The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low AN content in the plant as AN is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches for improving the yield of AN. In this work, the AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently the amount of AN is significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a greater number of trichomes. In order to study the role of AaMYB1 on trichome development and other possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. To support our findings in Arabidopsis thaliana, an AaMYB1 orthologue from this model plant, AtMYB61, was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA9 into the bioactive GA4 as well as the enzymes involved in the degradation of GA4 . Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connects important biosynthetic processes, and reveal its potential value for AN production through genetic engineering.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Artemisia annua/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Artemisia annua/genética , Giberelinas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/genética , Tricomas/genética , Tricomas/metabolismoRESUMO
Our lack of full understanding of transport and sequestration of the heterologous products currently limit metabolic engineering in plants for the production of high value terpenes. For instance, although all genes of the artemisinin/arteannuin B (AN/AB) biosynthesis pathway (AN-PW) from Artemisia annua have been identified, ectopic expression of these genes in Nicotiana benthamiana yielded mostly glycosylated pathway intermediates and only very little free (dihydro)artemisinic acid [(DH)AA]. Here we demonstrate that Lipid Transfer Protein 3 (AaLTP3) and the transporter Pleiotropic Drug Resistance 2 (AaPDR2) from A. annua enhance accumulation of (DH)AA in the apoplast of N. benthamiana leaves. Analysis of apoplast and cell content and apoplast exclusion assays show that AaLTP3 and AaPDR2 prevent reflux of (DH)AA from the apoplast back into the cells and enhances overall flux through the pathway. Moreover, AaLTP3 is stabilized in the presence of AN-PW activity and co-expression of AN-PW+AaLTP3+AaPDR2 genes yielded AN and AB in necrotic N. benthamiana leaves at 13 days post-agroinfiltration. This newly discovered function of LTPs opens up new possibilities for the engineering of biosynthesis pathways of high value terpenes in heterologous expression systems.
Assuntos
Artemisia annua/fisiologia , Artemisininas/metabolismo , Vias Biossintéticas/fisiologia , Proteínas de Transporte/metabolismo , Engenharia Metabólica/métodos , Nicotiana/fisiologia , Proteínas de Plantas/metabolismo , Artemisininas/isolamento & purificação , Proteínas de Transporte/genética , Melhoramento Genético/métodos , Redes e Vias Metabólicas/fisiologia , Proteínas de Plantas/genéticaRESUMO
The artemisinic aldehyde double bond reductase (DBR2) plays an important role in the biosynthesis of the antimalarial artemisinin in Artemisia annua. Artemisinic aldehyde is reduced into dihydroartemisinic aldehyde by DBR2. Artemisinic aldehyde can also be oxidized by amorpha-4,11-diene 12-hydroxylase and/or aldehyde dehydrogenase 1 to artemisinic acid, a precursor of arteannuin B. In order to better understand the effects of DBR2 expression on the flow of artemisinic aldehyde into either artemisinin or arteannuin B, we determined the content of dihydroartemisinic aldehyde, artemisinin, artemisinic acid and arteannuin B content of A. annua varieties sorted into two chemotypes. The high artemisinin producers (HAPs), which includes the '2/39', 'Chongqing' and 'Anamed' varieties, produce more artemisinin than arteannuin B; the low artemisinin producers (LAPs), which include the 'Meise', 'Iran#8', 'Iran#14', 'Iran#24' and 'Iran#47' varieties, produce more arteannuin B than artemisinin. Quantitative PCR showed that the relative expression of DBR2 was significantly higher in the HAP varieties. We cloned and sequenced the promoter of the DBR2 gene from varieties of both the LAP and the HAP groups. There were deletions/insertions in the region just upstream of the ATG start codon in the LAP varities, which might be the reason for the different promoter activities of the HAP and LAP varieties. The relevance of promoter variation, DBR2 expression levels and artemisinin biosynthesis capabilities are discussed and a selection method for HAP varieties with a DNA marker is suggested. Furthermore, putative cis-acting regulatory elements differ between the HAP and LAP varieties.
Assuntos
Artemisia annua/genética , Artemisia annua/metabolismo , Artemisininas/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antimaláricos/metabolismo , Artemisia annua/classificação , Sequência de Bases , DNA de Plantas/genética , Genes de Plantas , Plantas Medicinais/classificação , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Especificidade da EspécieRESUMO
Artemisinin derivatives are effective anti-malarial drugs. In order to design transgenic plants of Artemisia annua with enhanced biosynthesis of artemisinin, we are studying the promoters of genes encoding enzymes involved in artemisinin biosynthesis. A 1,151 bp promoter region of the cyp71av1 gene, encoding amorpha-4,11-diene 12-hydroxylase, was cloned. Alignment of the cloned promoter and other cyp71av1 promoter sequences indicated that the cyp71av1 promoter may be different in different A. annua varieties. Comparison to the promoter of amorpha-4,11-diene synthase gene showed a number of putative cis-acting regulatory elements in common, suggesting a co-regulation of the two genes. The cyp71av1 promoter sequence was fused to the ß-glucuronidase (GUS) reporter gene and two varieties of A. annua and Nicotiana tabacum were transformed. In A. annua, GUS expression was exclusively localized to glandular secretory trichomes (GSTs) of leaf primordia and top expanded leaves. In older leaves, there is a shift of expression to T-shaped trichomes (TSTs). Only TSTs showed GUS staining in lower leaves and there is no GUS staining in old leaves. GUS expression in flower buds was specifically localized to GSTs. The recombinant promoter carries the cis-acting regulatory elements required for GST-specific expression. The cyp71av1 promoter shows activity in young tissues. The recombinant promoter was up to 200 times more active than the wild type promoter. GUS expression in transgenic N. tabacum was localized to glandular heads. Transcript levels were up-regulated by MeJA. Wound responsiveness experiment showed that the cyp71av1 promoter does not appear to play any role in the response of A. annua to mechanical stress.
Assuntos
Artemisia annua/genética , Artemisia annua/metabolismo , Artemisininas/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antimaláricos/metabolismo , Sequência de Bases , DNA de Plantas/genética , Expressão Gênica , Genes de Plantas , Glucuronidase/genética , Glucuronidase/metabolismo , Redes e Vias Metabólicas , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Sesquiterpenos Policíclicos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido Nucleico , Sesquiterpenos/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Secondary attraction to aggregation pheromones plays a central role in the host colonization behavior of the European spruce bark beetle Ips typographus. However, it is largely unknown how the beetles pioneering an attack locate suitable host trees, and eventually accept or reject them. To find possible biomarkers for host choice by I. typographus, we analyzed the chemistry of 58 Norway spruce (Picea abies) trees that were subsequently either (1) successfully attacked and killed, (2) unsuccessfully attacked, or (3) left unattacked. The trees were sampled before the main beetle flight in a natural Norway spruce-dominated forest. No pheromones were used to attract beetles to the experimental trees. To test the trees' defense potential, each tree was treated in a local area with the defense hormone methyl jasmonate (MeJ), and treated and untreated bark were analyzed for 66 different compounds, including terpenes, phenolics and alkaloids. The chemistry of MeJ-treated bark correlated strongly with the success of I. typographus attack, revealing major chemical differences between killed trees and unsuccessfully attacked trees. Surviving trees produced significantly higher amounts of most of the 39 analyzed mono-, sesqui-, and diterpenes and of 4 of 20 phenolics. Alkaloids showed no clear pattern. Differences in untreated bark were less pronounced, where only 1,8-cineole and (-)-limonene were significantly higher in unsuccessfully attacked trees. Our results show that the potential of individual P. abies trees for inducing defense compounds upon I. typographus attack may partly determine tree resistance to this bark beetle by inhibiting its mass attack.
Assuntos
Adaptação Fisiológica , Picea/química , Plantas Comestíveis/química , Animais , Biomarcadores/análise , Besouros , Herbivoria , Fenóis/metabolismo , Feromônios/análise , Picea/crescimento & desenvolvimento , Casca de Planta/química , Sobrevida , Terpenos/metabolismoRESUMO
UNLABELLED: Artemisia annua L. produces a number of sesquiterpene synthases, which catalyze the conversion of farnesyl diphosphate to various sesquiterpenes. The cDNAs encoding amorpha-4,11-diene synthase (ADS), a key enzyme in the artemisinin biosynthesis, and epi-cedrol synthase (ECS), a complex sesquiterpene cyclization synthase, were cloned into Cowpea mosaic virus-based viral vector (pEAQ-HT) with Kozak consensus motif and C-terminal histidine tag. The plasmids were transformed into Agrobacterium LBA4404 and, agroinfiltrated into Nicotiana benthamiana leaves along with vector (pJL3:p19) containing Tomato bushy stunt virus post-transcriptional gene silencing suppressor. Quantitative PCR was carried out to measure the transcript levels at 0, 3, 6, 9, 12 and 15 days post-infiltration (dpi). The highest relative expression was observed at 9 dpi for both genes. Transiently expressed recombinant proteins of ADS and ECS were confirmed by SDS-PAGE and western blot. Recombinant proteins were extracted from 9 dpi leaves and purified by immobilized metal ion affinity chromatography using histidine tag, which produced yields of 90 and 96 mg kg⻹ fresh weight of leaves for ADS and ECS, respectively. Activities of the purified enzymes were assayed using gas chromatography-mass spectrometry for product identification and quantification using valencene as internal standard. The recombinant ADS and ECS converted farnesyl diphosphate into amorpha-4,11-diene (97 %) and epi-cedrol (96 %) as the major products, respectively. The purified enzymes exhibited the specific activity of 0.002 and 0.01 µmol min⻹ mg⻹ protein for ADS and ECS, respectively. The apparent k(cat) values were 2.1 × 10⻳ s⻹ and 11 × 10⻳ s⻹ for ADS and ECS, respectively. KEY MESSAGE: Agroinfiltration of leaves of Nicotiana bentamiana can be used to produce recombinant biosynthetic enzymes as exemplified by two sesquiterpene synthases from Artemisia annua in relatively high yields.
Assuntos
Alquil e Aril Transferases/biossíntese , Artemisia annua/enzimologia , Nicotiana/metabolismo , Proteínas de Plantas/biossíntese , Agrobacterium , Alquil e Aril Transferases/genética , Artemisia annua/genética , Clonagem Molecular , Vetores Genéticos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Sesquiterpenos/metabolismo , Nicotiana/genéticaRESUMO
BACKGROUND: Recently, Artemisia annua L. (annual or sweet wormwood) has received increasing attention due to the fact that the plant produces the sesquiterpenoid endoperoxide artemisinin, which today is widely used for treatment of malaria. The plant produces relatively small amounts of artemisinin and a worldwide shortage of the drug has led to intense research in order to increase the yield of artemisinin. In order to improve our understanding of terpene metabolism in the plant and to evaluate the competition for precursors, which may influence the yield of artemisinin, we have used qPCR to estimate the expression of 14 genes of terpene metabolism in different tissues. RESULTS: The four genes of the artemisinin biosynthetic pathway (amorpha-4,11-diene synthase, amorphadiene-12-hydroxylase, artemisinic aldehyde ∆11(13) reductase and aldehyde dehydrogenase 1) showed remarkably higher expression (between ~40- to ~500-fold) in flower buds and young leaves compared to other tissues (old leaves, stems, roots, hairy root cultures). Further, dihydroartemisinic aldehyde reductase showed a very high expression only in hairy root cultures. Germacrene A and caryophyllene synthase were mostly expressed in young leaves and flower buds while epi-cedrol synthase was highly expressed in old leaves. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase exhibited lower expression in old leaves compared to other tissues. Farnesyldiphosphate synthase, squalene synthase, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase showed only modest variation in expression in the different tissues, while expression of 1-deoxy-D-xylulose-5-phosphate synthase was 7-8-fold higher in flower buds and young leaves compared to old leaves. CONCLUSIONS: Four genes of artemisinin biosynthesis were highly expressed in flower buds and young leaves (tissues showing a high density of glandular trichomes). The expression of dihydroartemisinic aldehyde reductase has been suggested to have a negative effect on artemisinin production through reduction of dihydroartemisinic aldehyde to dihydroartemisinic alcohol. However, our results show that this enzyme is expressed only at low levels in tissues producing artemisinin and consequently its effect on artemisinin production may be limited. Finally, squalene synthase but not other sesquiterpene synthases appears to be a significant competitor for farnesyl diphosphate in artemisinin-producing tissues.
Assuntos
Artemisia annua/genética , Artemisininas/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Família Aldeído Desidrogenase 1 , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Artemisia annua/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Oxirredutases/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismoRESUMO
The sesquiterpene ß-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, ß-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of ß-caryophyllene. In this study, a full-length cDNA of a new duplicated ß-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce ß-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt-nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg-1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce ß-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography-mass spectrometry (GC-MS). GC-MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of ß-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.
RESUMO
New molecularly imprinted polymers (MIPs), which exhibit specific recognition of ethyl carbamate (EC) have been synthesized and studied. In this process, EC was the template molecule and ß-cyclodextrin derivatives were employed as functional monomers in the molecular imprinting technique (MIT). An EC molecularly imprinted sensor (EC-MIS) was prepared by using MIT surface modification. The EC-MIS was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. EC detection performance, binding parameters and dynamics mechanism were investigated. The result showed that the synthetic route designed was appropriate and that new MIP and EC-MIS were successfully prepared. The EC-MIS exhibited a good molecular recognition of EC. A linear relationship between current and EC concentration was observed using cyclic voltammetry and the detection limit was 5.86 µg L-1. The binding constant (K = 4.75 × 106 L mol-1) between EC and the EC-MIS, as well as, the number of binding sites (n = 1.48) has been determined. The EC-MIS recognition mechanism for the EC is a two-step process. The sensor was applied for the determination of EC in Chinese yellow wines, and the results were in good agreement with the gas chromatography-mass spectrometry (GC-MS) method.
RESUMO
Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.
Assuntos
Artemisia annua/genética , Artemisia annua/metabolismo , Artemisininas/metabolismo , Evolução Molecular , Genes de Plantas/genética , Genômica , Engenharia Metabólica , Anotação de Sequência MolecularRESUMO
The binding properties of pentacyclic triterpenoid isomeric drugs, i.e. ursolic acid (UA) and oleanolic acid (OA), to bovine lactoferrin (BLF) have been studied by molecule modeling, fluorescence spectroscopy, UV-visible absorbance spectroscopy and infrared spectroscopy (IR). Molecular docking, performed to reveal the possible binding mode or mechanism, suggested that hydrophobic interaction and hydrogen bonding play important roles to stabilize the complex. The results of spectroscopic measurements showed that the two isomeric drugs both strongly quenched the intrinsic fluorescence of BLF through a static quenching procedure although some differences between UA and OA binding strength and non-radiation energy transfer occurred within the molecules. The number of binding sites was 3.44 and 3.10 for UA and OA, respectively, and the efficiency of Förster energy transfer provided a distance of 0.77 and 1.21nm for UA and OA, respectively. The conformation transformation of BLF affected by the drugs conformed to the "all-or-none" pattern. In addition, the changes of the ratios of α-helices, ß-sheets and ß-turns of BLF during the process of the interaction were obtained. The results of the experiments in combination with the calculations showed that there are two modes of pentacyclic triterpenoid binding to BLF instead of one binding mode only governed by the principle of the lowest bonding energy.
Assuntos
Lactoferrina/metabolismo , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Triterpenos/química , Triterpenos/metabolismo , Animais , Sítios de Ligação , Bovinos , Transferência de Energia , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Isomerismo , Lactoferrina/química , Metais/química , Metais/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Triptofano/química , Triptofano/metabolismo , Ácido UrsólicoRESUMO
In order to obtain the biological active compound, α-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of α-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of α-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (1H NMR and 13C NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained α-mangostin was 95.6% as determined by HPLC analysis. The binding of native α-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that α-mangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to α-mangostin is 6.4832×105 L/mol and 1.4652×105 L/mol at 298 K and 7.8619×105 L/mol and 1.1582×105 L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between α-mangostin and HSA or TRF were also obtained by virtue of the Förster theory of non-radiation energy transfer. The effect of α-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between α-mangostin and HSA is hydrophobic interactions, while the main interaction between α-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.
Assuntos
Garcinia mangostana/química , Albumina Sérica/metabolismo , Transferrina/metabolismo , Xantonas/isolamento & purificação , Xantonas/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Albumina Sérica/química , Xantonas/farmacologiaRESUMO
A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-beta-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI=5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, beta-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K(m)- and k(cat)-values for farnesyl diphosphate, is 2.1 microM and 9.5 x 10(-3) s(-1), respectively resulting in the efficiency 4.5 x 10(-3) M(-1)s(-1). The enzyme exhibits substantial activity in the presence of Mg(2+), Mn(2+) or Co(2+) but essentially no activity when Zn(2+), Ni(2+) or Cu(2+) is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 microM for Mg(2+), Co(2+) or Mn(2+), respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.
Assuntos
Artemisia annua/enzimologia , Pirofosfatases/metabolismo , Proteínas Recombinantes/metabolismo , Artemisia annua/metabolismo , Cátions , Expressão Gênica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pirofosfatases/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off-gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on-line.
Assuntos
Técnicas Biossensoriais , Morinda/citologia , Nicotiana/citologia , Divisão Celular , Linhagem Celular , Redes Neurais de ComputaçãoRESUMO
The effective anti-malarial medicine artemisinin is costly because of the low content in Artemisia annua. Genetic engineering of A. annua is one of the most promising approaches to improve the yield of artemisinin. In this work, the transcription factor AaWRKY1, which is thought to be involved in the regulation of artemisinin biosynthesis, was cloned from A. annua var. Chongqing and overexpressed using the CaMV35S promoter or the trichome-specific CYP71AV1 promoter in stably transformed A. annua plants. The transcript level of AaWRKY1 was increased more than one hundred times under the CaMV35S promoter and about 40 times under the CYP71AV1 promoter. The overexpressed AaWRKY1 activated the transcription of CYP71AV1 and moreover the trichome-specific overexpression of AaWRKY1 improved the transcription of CYP71AV1 much more effectively than the constitutive overexpression of AaWRKY1, i.e. up to 33 times as compared to the wild-type plant. However the transcription levels of FDS, ADS, and DBR2 did not change significantly in transgenic plants. The significantly up-regulated CYP71AV1 promoted artemisinin biosynthesis, i.e. up to about 1.8 times as compared to the wild-type plant. It is demonstrated that trichome-specific overexpression of AaWRKY1 can significantly activate the transcription of CYP71AV1 and the up-regulated CYP71AV1 promotes artemisinin biosynthesis.
Assuntos
Alquil e Aril Transferases/genética , Artemisia annua/metabolismo , Artemisininas/metabolismo , Fatores de Transcrição/genética , Alquil e Aril Transferases/metabolismo , Sequência de Aminoácidos , Artemisia annua/genética , Perfilação da Expressão Gênica , Estrutura Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Fatores de Transcrição/metabolismoRESUMO
Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to ß-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.
Assuntos
Artemisia annua/enzimologia , Hidroliases/genética , Acetatos , Artemisia annua/genética , Sequência de Bases , Ciclopentanos , Glucuronidase/genética , Hidroliases/metabolismo , Dados de Sequência Molecular , Oxilipinas , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Transformação GenéticaRESUMO
In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for ß-caryophyllene (CPS), epi-cedrol (ECS) and ß-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.
Assuntos
Alquil e Aril Transferases/genética , Artemisia annua/enzimologia , Artemisia annua/genética , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Regiões Promotoras Genéticas , Sesquiterpenos/metabolismo , Acetatos/farmacologia , Alquil e Aril Transferases/metabolismo , Artemisia annua/efeitos dos fármacos , Sequência de Bases , Southern Blotting , Ciclopentanos/farmacologia , Flores/efeitos dos fármacos , Flores/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucuronidase/metabolismo , Oxilipinas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Sesquiterpenos/química , Especificidade por Substrato/efeitos dos fármacosRESUMO
The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.