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Metabolic Engineering of the Feverfew Parthenolide Pathway in N. Benthamiana

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Metabolic Engineering of the Feverfew Parthenolide Pathway in N. Benthamiana

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Samenvatting

Fragments of cytochrome P450 genes (P450s), lipid transfer protein genes (LTPs) and ATP-binding cassette subfamily G transporter genes (ABCGs) were amplified from cDNA of feverfew (Tanacetum parhenium) ovary phase 4 RNA using PCR. P450 gene fragments were cloned into ImpactTim entry vector using the restriction enzymes Not1 and Pac1 and T4 DNA ligase. LTP-12309 and LTP-21667 gene fragments were cloned into TOPO TA entry vector. LTP-14333, LTP-19412, ABCg-3885 and ABCg-7696 gene fragments were cloned into D-TOPO entry vector.
The following positive entry clones (from E. coli) were used for cloning into destination vector via Gateway Technology LR Clonase 2: P450-8272-E1, P450-8595-E4, P450-9025-E2, LTP-14333-E8, LTP-19412-E4, LTP-21667-E2 and ABCg-3885-E5. P450 gene fragments were cloned into pBinPlus vector and LTP- and ABCg-3885 gene fragments were cloned into pB7GW2 vector. The following destination clones were successfully transformed from E. coli into A. tumefaciens: P450-8272-D1, P450-8595-D3, P450-9025-D4, LTP-14333-D5, LTP-19412-D2, LTP-21667-D3 and ABCg-3885-D1.
A. tumefaciens cultures containing P450-8595-D3-2 and P450-9025-D4-2 destination clones, together with costunolide pathway genes, were infiltrated in N. benthamiana leafs. Leaf extracts were analysed using LC-QTOF-MS. Unfortunately, no conclusion can be given about the effect of the agro-infiltration treatments. This is due to the fact that some samples were mixed up. Nevertheless, no derivative compound of costunolide was detected.

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OrganisatieAvans Hogeschool
OpleidingBiologie en Medisch Laboratoriumonderzoek-Breda
AfdelingATGM Academie voor de technologie van Gezondheid en Milieu
PartnerWageningen University and Research Centre
Datum2014-06-12
TypeBachelor
TaalEngels

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