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Team:Valencia UPV/Project/results/trichome expression
From 2014.igem.org
Project > Results > Trichome-Specific Expression
This project main objective was to produce insect sexual pheromones in genetically engineered plants, but once this goal was achieved, another important question raised, would that plant be able to release these pheromones into the environment.
Therefore, mimicking nature, the chosen approach was to express the enzymes of the pheromone production pathway (see biosynthesis) specifically on glandular trichomes, which are plant organs, specialized on releasing volatiles. With this objective, a glandular trichome-specific promoter (PCPS2) was obtained from Nicotiana tabacum genome for further use. (see Module: Pheromone release)
First of all, the promoter needed to be tested. PCPS2 promoter was joined with GFP in order to look for localized fluorescence in the trichomes. (see GoldenBraid 2.0)
Figure 1. Genetic construct of the Glandular trichomes specific promoter PCPS2 and GFP.
This construct was inserted into the plant, Nicotiana benthamiana (see agroinfiltration) and after 5 days the expression of GFP was checked. We used a Leica Stereo-Fluorescence microscope using a GFP2 filter (460-500nm >510 LP) After exciting the sample at a 395nm wavelength, regions expressing GFP showed a bright light green colour (Figure 2).
This is what was observed:
Figure 2. Glandular trichomes specific GFP expression analysis.
Figure 3. Negative control showing no glandular trichomes specific GFP expression.
As it can be seen, GFP is specifically expressed on glandular trichomes. This proves that this promoter works and it can be used to express the enzymes of the pheromone production pathway specifically in them.
With the selective expression of the production pathway, the plant will be able to improve the pheromone release since the trichomes are specialized organs; and also reduce the metabolic cost of the pathway, compared with the production in the whole plant using a constitutive promoter e.g. P35S.
Now that the PCPS2 promoter has proven to work it had to be assembled with the actual genes of interest, the enzymes of the insect sexual pheromone pathway. (See Results: Constructs: Release)
