Grants and Contracts Details
Description
During the initial 18-month funding period for the DOE-ARPA-E TAMU grant, the Chappell
laboratory will be responsible for 5 research objectives:
1. The Chappell laboratory will first identify all the known, functionally characterized
limonene and caryophyllene synthase genes to date and compile a list of their known
biochemical features. These will include kinetic constants and reaction product
specificity. From this initial list of gene candidates, the 3 best limonene and
caryophyllene synthases as defined by their catalytic efficiency (kcat/Km) and reaction
product specificity will be prioritized for further confirmation studies.
2. The Chappell laboratory will verify these biochemical characteristics experimentally for 3
limonene synthase and caryophyllene synthases. The genes encoding for these
enzymes will be obtained by either direct cloning from their native hosts, or synthetic
forms of the genes will be purchased from a private vendor. The genes will be
incorporated into suitable bacterial expression vectors and the heterologous produced
enzymes purified based on affinity tags appended to the amino or carboxy terminal
regions of the genes. The purified proteins will then be used in standard terpene
synthase enzyme assays, the activity of the enzymes determined, their kinetic constants
calculated, and the profile of their reaction products determined by GC-MS.
3. In parallel to the biochemical assessments of these terpene synthase genes, the
selected terpene synthase genes will be inserted into Ti-plasmid vectors for engineering
these constructs into transgenic tobacco plants. The Ti-plasmid vectors will be similar to
those described by Wu et al. (2006 Nature Biotech. 24:1441) but modified to effect
constitutive and trichome specific gene expression with the encoded proteins targeted to
either the cytosolic or chloroplast compartments. The various Ti-plasmid vectors will be
inserted into Agrobacterium tumefaciens and these bacteria used to introduce the
various gene constructs into transgenic tobacco lines as described by Wu et al. (2006
Nature Biotech. 24:1441). Twenty independent transgenic tobacco lines for each
construct will be generated in this manner and the T0 generation propagated in the
greenhouse and screened for their limonene and caryophyllene levels by GC-MS
analysis.
4. The Chappell laboratory will also participate with other investigators in this project to
perform genetic crosses between the various tobacco lines developed during the course
of this project, generating hybrid lines. We will reposit these seed lines and distribute
these seed lines to the various participating research groups as requested.
5. The Chappell laboratory will also perform field tests of select transgenic lines at the UK
Spindle Top research farm. Typically experimental tests consisting of plants grown in
triplicate settings and evaluating agronomic (biomass accumulation, high, leaf area) and
physiological (photosynthetic measurements) performance, along with determining
terpene chemical composition by GC-MS of tissue sample extracts are anticipated.
Status | Finished |
---|---|
Effective start/end date | 2/15/12 → 8/14/13 |
Funding
- Texas AgriLife Research: $232,210.00
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