RESUMEN
Via studying the phenotype, growth curve and secondary metabolites of two kinds of suspension culture cell of Arnebia euchroma, the kinetics parameters of growth and accumulation of shikonin compounds in cell suspension culture of A. euchroma was obtained through simulating and modeling. This Study found that the red high-yielding one was a fine cell line for producing shikonin compounds, and the white low-yielding one may be a mutant. The first-order and second-order derivative of the fitting function were obtained by fitting the Logistic model of growth curve to get the growth rate and growth acceleration curve of the suspended cells. It is found that the best period to subculture was the 15th day cultured in fresh medium, and the best period of the induction process was the 13th-14th day. When compared the growth rate of the red line and the shikonin compounds accumulation curve, it is found that the rapid growth of the biomass of cells was not conducive to the synthesis and accumulation of shikonin compounds.
Asunto(s)
Boraginaceae/citología , Boraginaceae/metabolismo , Naftoquinonas/metabolismo , Boraginaceae/química , Técnicas de Cultivo de Célula , Proliferación Celular , Células VegetalesRESUMEN
Artemisinin is an effective antimalarial drug isolated from the medicinal plant Artemisia annua L. Due to its increasing market demand and the low yield in A. annua, there is a great interest in increasing its production. In this paper, in an attempt to increase artemisinin content of A. ANNUA by suppressing the expression of ß-caryophyllene synthase, a sesquiterpene synthase competing as a precursor of artemisinin, the antisense fragment (750 bp) of ß-caryophyllene synthase cDNA was inserted into the plant expression vector pBI121 and introduced into A. annua by Agrobacterium-mediated transformation. PCR and Southern hybridization confirmed the stable integration of multiple copies of the transgene in 5 different transgenic lines of A. annua. Reverse transcription PCR showed that the expression of endogenous CPS in the transgenic lines was significantly lower than that in the wild-type control A. annua plants, and ß-caryophyllene content decreased sharply in the transgenic lines in comparison to the control. The artemisinin content of one of the transgenic lines showed an increase of 54.9â% compared with the wild-type control. The present study demonstrated that the inhibition pathway in the precursor competition for artemisinin biosynthesis by anti-sense technology is an effective means of increasing the artemisinin content of A. annua plants.
Asunto(s)
Antiinfecciosos/metabolismo , Artemisia annua/metabolismo , Artemisininas/metabolismo , Medicamentos Herbarios Chinos/metabolismo , Ligasas/genética , Antiinflamatorios no Esteroideos/metabolismo , Artemisia annua/enzimología , Artemisia annua/genética , ADN sin Sentido/genética , ADN Complementario/genética , ADN de Plantas/genética , Regulación hacia Abajo/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Medicina Tradicional China , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotes de la Planta/enzimología , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Plantas Medicinales , Plásmidos , Sesquiterpenos Policíclicos , ARN Mensajero/genética , ARN de Planta/genética , Plantones/enzimología , Plantones/genética , Plantones/metabolismo , Sesquiterpenos/metabolismoRESUMEN
This paper provides evidence that salicylic acid (SA) can activate artemisinin biosynthesis in Artemisia annua L. Exogenous application of SA to A. annua leaves was followed by a burst of reactive oxygen species (ROS) and the conversion of dihydroartemisinic acid into artemisinin. In the 24 h after application, SA application led to a gradual increase in the expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene and a temporary peak in the expression of the amorpha-4,11-diene synthase (ADS) gene. However, the expression of the farnesyl diphosphate synthase (FDS) gene and the cytochrome P450 monooxygenase (CYP71AV1) gene showed little change. At 96 h after SA (1.0 mM) treatment, the concentration of artemisinin, artemisinic acid and dihydroartemisinic acid were 54, 127 and 72% higher than that of the control, respectively. Taken together, these results suggest that SA induces artemisinin biosynthesis in at least two ways: by increasing the conversion of dihydroartemisinic acid into artemisinin caused by the burst of ROS, and by up-regulating the expression of genes involved in artemisinin biosynthesis.
Asunto(s)
Artemisia annua/enzimología , Artemisininas/metabolismo , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacología , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Artemisia annua/efectos de los fármacos , Artemisia annua/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Geraniltranstransferasa/genética , Geraniltranstransferasa/metabolismo , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/genética , Hidroximetilglutaril-CoA-Reductasas NADP-Dependientes/metabolismo , Estructura Molecular , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Benzalacetone synthase (BAS) is a member of the plant-specific type III PKS superfamily that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce p-hydroxybenzalacetone. In our recent work (Ma et al. in Planta 229(3):457-469, 2008), a three-intron type III PKS gene (PcPKS2) was isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed this recombinant PcPKS2 to be a BAS. In this study, another three-intron type III PKS gene (PcPKS1) and its corresponding cDNA were isolated from P. cuspidatum. Sequence and phylogenetic analyses demonstrated that PcPKS1 is a chalcone sythase (CHS). However, functional and enzymatic analyses showed that recombinant PcPKS1 is a bifunctional enzyme with both, CHS and BAS activity. DNA gel blot analysis indicated that there are two to four CHS copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS1 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS1 transcripts in leaves were inducible by pathogen infection and wounding. BAS is thought to play a crucial role in the construction of the C(6)-C(4) moiety found in a variety of phenylbutanoids, yet so far phenylbutanoids have not been isolated from P. cuspidatum. However, since PcPKS1 and PcPKS2 (Ma et al. in Planta 229(3):457-469, 2008) have been identified in P. cuspidatum, it is possible that such compounds are also produced in that plant, albeit in low concentrations.
Asunto(s)
Acetona/metabolismo , Aciltransferasas/genética , Fallopia japonica/enzimología , Fallopia japonica/genética , Flavanonas/biosíntesis , Genes de Plantas , Intrones/genética , Acetona/química , Aciltransferasas/química , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Flavanonas/química , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/metabolismo , Análisis de Secuencia , Especificidad por SustratoRESUMEN
A type III polyketide synthase cDNA and the corresponding gene (PcPKS2) were cloned from Polygonum cuspidatum Sieb. et Zucc. Sequencing results showed that the ORF of PcPKS2 was interrupted by three introns, which was an unexpected finding because all type III PKS genes studied so far contained only one intron at a conserved site in flowering plants, except for an Antirrhinum majus chalcone synthase gene. Besides the unusual gene structure, PcPKS2 showed some interesting characteristics: (1) the CHS "gatekeepers" Phe215 and Phe265 are uniquely replaced by Leu and Cys, respectively; (2) recombinant PcPKS2 overexpressed in Escherichia coli efficiently afforded 4-coumaroyltriacetic acid lactone (CTAL) as a major product along with bis-noryangonin (BNY) and p-hydroxybenzalacetone at low pH; however, it effectively yielded p-hydroxybenzalacetone as a dominant product along with CTAL and BNY at high pH. Beside p-hydroxybenzalacetone, CTAL and BNY, a trace amount of naringenin chalcone could be detected in assays at different pH. Furthermore, 4-coumaroyl-CoA and feruloyl-CoA were the only cinnamoyl-CoA derivatives accepted as starter substrates. PcPKS2 did not accept isobutyryl-CoA, isovaleryl-CoA or acetyl-CoA as substrate. DNA gel blot analysis indicated that there are two to four PcPKS2 copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS2 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS2 transcripts in leaves were induced by pathogen infection, but not by wounding.
Asunto(s)
Fallopia japonica/enzimología , Genes de Plantas , Proteínas de Plantas/genética , Sintasas Poliquetidas/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Fallopia japonica/genética , Expresión Génica , Intrones , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Sintasas Poliquetidas/química , Sintasas Poliquetidas/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Terpenoids are present in all organisms but are especially abundant in plants, with more than 30,000 compounds. Not only do they play an important role in the life of plant, but also have high commercial values. However, the content of many important terpenoids in plant is very low. Therefore, how to improve the inefficient production of terpenoids is an urgent task. Metabolic engineering has been one of the most potential technologies to improve terpenoids production in recent years, following the study of metabolic pathway and regulation mechanism of terpenoids. Although there are some breakthroughs, metabolic engineering of terpenoids is still full of challenges because of the lack of knowledge on metabolic control of most terpenoids. Functional genomics approaches, including transcriptomics, proteomics and metabolomics, are potential tools for exploring of metabolic engineering. Integrating transcriptomics and metabolomics is an effective way to discover new genes involved in metabolic pathway. In this paper, the representative research outcomes about the metabolic engineering of terpenoids in plant were reviewed concisely and then the application of functional genomics approaches to study metabolic pathway and regulation mechanism of terpenoids and the strategies for metabolic engineering of terpenoids were discussed.
Asunto(s)
Plantas/metabolismo , Ingeniería de Proteínas/métodos , Terpenos/metabolismo , Genómica/métodos , Metabolómica/métodos , Proteómica/métodosRESUMEN
Salidroside is a novel effective adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Because this plant is a rare resource and has low yield, there is great interest in enhancing the production of salidroside. In this study, a putative UDP-glucosyltransferase (UGT) cDNA, UGT73B6, was isolated from Rhodiola sachalinensis using a rapid amplification of cDNA ends (RACE) method. The cDNA was 1,598 bp in length encoding 480 deduced amino acid residues with a conserved UDP-glucose-binding domain (PSPG box). Southern blot analysis of genomic DNA indicated that UGT73B6 existed as a single copy gene in the R. sachalinensis genome. Northern blot analysis revealed that transcripts of UGT73B6 were present in roots, calli and stems, but not in leaves. The UGT73B6 under 35S promoter with double-enhancer sequences from CaMV-Omega and TMV-Omega fragments was transferred into R. sachalinensis via Agrobacterium tumefaciens. PCR, PCR-Southern and Southern blot analyses confirmed that the UGT73B6 gene had been integrated into the genome of transgenic calli and plants. Northern blot analysis revealed that the UGT73B6 gene had been expressed at the transcriptional level. High performance liquid chromatography (HPLC) analysis indicated that the overexpression of the UGT73B6 gene resulted in an evident increase of salidroside content. These data suggest that the cloned UGT73B6 can regulate the conversion of tyrosol aglycon to salidroside in R. sachalinensis. This is the first cloned glucosyltransferase gene involved in salidroside biosynthesis.
Asunto(s)
Glucósidos/biosíntesis , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Rhodiola/genética , Rhodiola/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Glucósidos/química , Glucósidos/genética , Glucosiltransferasas/química , Datos de Secuencia Molecular , Estructura Molecular , Fenoles/química , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMEN
Artemisinin,a new and a very potent antimalarial drug, is produced by the plant Artemisia annua L. with a very low yield ranging from 0.01% to 0.8% on a dry-weight basis. This makes artemisinin an expensive drug. Several studies reported chemical synthesis of the artemisinin, but none of them seems a viable economical alternative compared with the isolation of artemisinin from the plant. Hence, a higher artemisinin concentration in the plant is necessary for cheap antimalarial drug production. Many types of cyclic sesquiterpenes in Artemisia annua have been characterized to date, each derived from the common cyclic precursor FDP in a reaction catalyzed by a sesquiterpene synthase. Sesquiterpene synthases are widely regarded as the rate-determining regulatory enzymes in the pathways they participate, and a number of sesquiterpene synthases have been cloned from Artemisia annua up to now. This report is a brief review on the following sesquiterpene synthases: epi-cedrol synthase, amorpha-4,11-diene synthase, beta-caryophyllene synthase, (E)-beta-farnesene synthase, germacrene A synthase, as well as a new sesquiterpene synthase whose function remains largely unknown. The report is of help for a better understanding of metabolic engineering of Artemisia annua.
Asunto(s)
Transferasas Alquil y Aril/genética , Artemisia annua/enzimología , Artemisininas/metabolismo , Liasas de Carbono-Carbono/genética , Transferasas Alquil y Aril/biosíntesis , Secuencia de Aminoácidos , Antimaláricos , Artemisia annua/genética , Liasas de Carbono-Carbono/biosíntesis , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Sesquiterpenos/aislamiento & purificaciónRESUMEN
The flowering promoting factor1 ( fpf1) from Arabidopsis thaliana was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The fpf1 gene was firstly inserted in the binary vector pBI121 under the control of CaMV 35S promoter to construct the plant expression vector pBIfpf1, then leaf explants of A. annua were infected with A. tumefaciens LBA4404 containing pBIfpf1, and induced shoots. Transgenic plants were obtained through the selection with kanamycin. PCR, PCR-Southern and Southern blot analyses confirmed that the foreign fpf1 gene had been integrated into the A. annua genome. RT-PCR and RT-PCR-Southern analyses suggested that the foreign fpf1 gene had expressed at the transcriptional level. Under short-day conditions, the flowering time of fpf1 transgenic plants was about 20 days earlier than the non-transformed plants; however, no significant differences were detected in artemisinin content between the flowering transgenic plants and the non-flowering non-transgenic plants. These results showed that flowering is not a necessary factor for increasing the artemisinin content, furthermore, there may be no direct linkage between flowering and artemisinin biosynthesis.
Asunto(s)
Proteínas de Arabidopsis/genética , Artemisia annua/genética , Artemisininas/metabolismo , Fitoterapia , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo , Artemisia annua/crecimiento & desarrollo , Artemisia annua/metabolismo , Cartilla de ADN , Flores , Humanos , Luz , Hojas de la Planta , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Artemisinin, a new and a very potent antimalarial drug, is produced by the Chinese medicinal herb Artemisia annua L. It is a sesquiterpene lactone with an endoperoxide bridge and is active against chloroquine resistant forms of Plasmodium falciparum. The relatively low yield (0.01% - 0.6%) of artemisinin in A. annua is a serious limitation to the commercialization of the drug. Therefore, a through understanding of the biosynthetic pathway and the characterization of the involved enzymes are important for the biology production of artemisinin. This review is focused on the recent progress in the molecular regulation of artemisinin biosynthesis from the following aspects: the biosynthetic pathway of artemisinin, the key enzymes involved in artemisinin biosynthesis, and the molecular regulation of artemisinin biosynthesis. The biosynthetic pathway of artemisinin belongs to the isoprenoid metabolite pathway, the key enzymes involved in the biosynthesis of artemisinin include: 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FDPS), and amorpha-4, 11-diene synthase, of which amorpha-4, 11-diene synthase catalyzes the cyclisation of the ubiquitous precursor farnesyl diphosphate to the highly specific olefinic sesquiter-pene skeletons and has been postulated as the regulatory step in the biosynthesis of artemisinin. Recently the gene encoding of the amorpha-4, 11-diene synthase has been cloned and the functional expressions have been studied by several research teams, therefore, the breakthroughs in production of artemisinin could hopefully be achieved by metabolic engineering of the plant, in particular, by over-expressing enzyme(s) catalyzing the rate limiting step(s) of artemisinin biosynthesis or by inhibiting the enzyme(s) of other pathway competing for its precursors. Besides, the effects of the heterogenesis isoprenoid pathway related genes on artemisinin biosynthesis of the transformed plants were also discussed.
Asunto(s)
Artemisininas/metabolismo , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Antimaláricos/metabolismo , Artemisia annua/enzimología , Artemisia annua/genética , Artemisia annua/metabolismo , Biotecnología/métodos , Modelos Biológicos , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
OBJECTIVE: To study the effect of several factors on the quantity of hypericin in H. perforatum callus. METHOD: High efficiency liquid phase chromatography and plant tissue culture were applied. RESULT AND CONCLUSION: When the ratio of nitro-nitrogen to amina-nitrogen is 3:1, the callus biomass is 1.6-fold and the synthetic mass of hypericin rises. 0.1-0.20 mg x L(-1) mannose improves the content of total hypericin. The addition of PVP or PVPP can promote improvement of the growth and biosynthesis of callus.