title: Genetic Transformation of Artemisia carvifolia Buch with rol Genes Enhances Artemisinin Accumulation.
abstract: The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01-0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism.
authors.affiliation: Department of Biochemistry, Faculty of Biological sciences, Quaid-i-Azam University, Islamabad, Pakistan.
authors.forename: Erum
authors.initials: E
authors.lastname: Dilshad
authors.affiliation: Laboratorio de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Spain.
authors.forename: Rosa Maria
authors.initials: RM
authors.lastname: Cusido
authors.affiliation: Laboratorio de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Spain.
authors.forename: Karla
authors.initials: K
authors.lastname: Ramirez Estrada
authors.affiliation: Laboratorio de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Bonfill
authors.affiliation: Department of Biochemistry, Faculty of Biological sciences, Quaid-i-Azam University, Islamabad, Pakistan.
authors.forename: Bushra
authors.initials: B
authors.lastname: Mirza
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000962:Antimalarials / Q000378:metabolism* / Q000600:supply & distribution; D000077549:Artemether; D031172:Artemisia annua / Q000235:genetics* / Q000378:metabolism*; D037621:Artemisinins / Q000378:metabolism* / Q000600:supply & distribution; D000077332:Artesunate; D001483:Base Sequence; D053898:Biosynthetic Pathways; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic; D064201:Trichomes / Q000235:genetics / Q000378:metabolism
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D010940:Plant Proteins; D000077332:Artesunate; C039060:artenimol; C031327:artemisinin; D000077549:Artemether