RESUMO
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.
Assuntos
Anti-Infecciosos/metabolismo , Artemisia annua/metabolismo , Artemisininas/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Ligases/genética , Anti-Inflamatórios não Esteroides/metabolismo , Artemisia annua/enzimologia , Artemisia annua/genética , DNA Antissenso/genética , DNA Complementar/genética , DNA de Plantas/genética , Regulação para Baixo/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Medicina Tradicional Chinesa , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotos de Planta/enzimologia , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Plantas Medicinais , Plasmídeos , Sesquiterpenos Policíclicos , RNA Mensageiro/genética , RNA de Plantas/genética , Plântula/enzimologia , Plântula/genética , Plântula/metabolismo , Sesquiterpenos/metabolismoRESUMO
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.
Assuntos
Artemisia annua/enzimologia , Artemisininas/metabolismo , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Artemisia annua/efeitos dos fármacos , Artemisia annua/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/genética , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/metabolismo , Estrutura Molecular , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.