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Team:Valencia UPV/Project/results/pheromone analysis
From 2014.igem.org
Project > Results > Pheromone Analysis
It was a long way to get here but, after our first results, future looked promising. This is what we got so far: the different parts needed to build the pheromone biosynthesis device were domesticated and assembled with the help of GoldenBraid 2.0. Once the constructs were obtained, they were transiently transformed by agroinfiltration into our plant chassis, N. benthamiana. Now, the moment of truth has arrived: do our plants actually produce the target pheromones?
To answer this question, we analysed the volatiles produced by our "Sexy Plants" using HS-SPME coupled to GC-MS. We co-infiltrated our pheromone biosynthesis device together with a construct carrying the silencing suppressor P19(*), as well as the P19 construct alone as negative control. Below you can see a representative full scan chromatogram of each of them.
Figure 1. GC-MS analysis of the volatile organic compounds from a genetically engineered and control N. benthamiana plants. On the right, an overlay chromatogram (control/sexy plant) of the two pheromone peaks from a SIM mode acquisition for pheromone representative ions.
With just one glance, we could identify two peaks in the genetically engineered N. benthamiana plants that were not present in the control.
Could those peaks be our pheromones? A comparison of their mass spectra with the NIST mass spectrum library retrieved (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate as best matches. Furthermore, this putative identification was confirmed by comparing their retention time and mass spectra with those of pure (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate synthesised at the CEQA (UPV, Spain) and analysed under identical GC-MS conditions. So, yes, our genetically engineered N. benthamiana is sexy!!!!
More good news. Not only were the pheromones being produced, but they were also two of the most abundant volatiles in the plant. In addition, the ratio between the abundance of (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate was approximately 4 to 1. This means that unprecedented conversion rates were achieved, compared to previous publications (Ding et al. 2014). Such superior conversion rate can be attributed to the use of a single multigene construction (see Results- Constructs- Biosynthesis) comprising all three genes needed to produce the pheromones in a single plasmid and thus, facilitating the simultaneous expression of the three enzymes in the plant cells (see Biosynthesis).
But wait, there's more! Our sexy plants had one surprise in store for us. Even though we did not succeeded in cloning FAO1, the enzyme catalyzing the the biosynthesis of (Z)-11-hexadecenal from (Z)-11-hexadecen-1-ol (see Notebook: FAO1 obtention), we could identify a small peak in our engineered plants chromatograms that was absent in the controls and had a mass spectrum that matched that of (Z)-11-hexadecenal. The analysis of a pure (Z)-11-hexadecenal standard (synthesised at the CEQA) confirmed our expectations, our sexy plants produce a small amount of (Z)-11-hexadecenal. How? We think that the conversion of (Z)-11-hexadecen-1-ol into (Z)-11–hexadecenal is probably performed by an endogenous alcohol oxidase from the plant, present in the genome of N. benthamiana but with not apparent physiological relevance (Sol Genomics Network). As the conversion rate was low, this opened a field for futher studies on pheromone plant-production.
Figure 3. GC-MS detection of (Z)-11-hexadecenal in N. benthamiana leaves .
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At the end of the journey we have achieved a plant able to produce three insect sexual pheromones:
- (Z)-11-hexadecen-1-ol as the most abundant plant volatile.
- (Z)-11-hexadecenyl acetate as one of the most abundant volatile molecules in the plant, with an unprecedented conversion yield.
- (Z)-11-hexadecenal, produced by an endogenous plant enzyme at low yield that could be improved in future studies.
