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Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana.
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
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2012 (English)In: Plant Cell Reports, ISSN 0721-7714, E-ISSN 1432-203X, Vol. 31, no 7, 1309-1319 p.Article in journal (Refereed) Published
Abstract [en]

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 syn- thase, 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 chroma- tography using histidine tag, which produced yields of 90 and 96 mg kg-1 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 con- verted 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 mmol min-1 mg-1 protein for ADS and ECS, respectively. The apparent kcat values were 2.1 x 10-3 s-1 and 11 x 10-3 s-1 for ADS and ECS, respectively.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2012. Vol. 31, no 7, 1309-1319 p.
National Category
Botany
Research subject
Chemistry, Biochemistry
Identifiers
URN: urn:nbn:se:lnu:diva-21154DOI: 10.1007/s00299-012-1250-zOAI: oai:DiVA.org:lnu-21154DiVA: diva2:544016
Available from: 2012-08-13 Created: 2012-08-13 Last updated: 2016-10-25Bibliographically approved

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Kanagarajan, SelvarajuMuthusamy, Sarala DeviGliszczynska, AnnaLundgren, AnneliBrodelius, Peter E.
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