Team:Valencia UPV/Project/results/constructs

Project > Results > Constructs

RESULTS – CONSTRUCTS- BIOSYNTHESIS

In order to engineer the insect sexual pheromone pathway in our Sexy plant, we had to isolate four genes from different organisms: a desaturase (AtrΔ11), a reductase (HarFAR), an acetyltransferase (EaDAcT) and finally an alcohol oxidase (FAO). As they were coming from very different and not easily accessible organisms (two moths and a plant from Asia), the coding sequences (CDS) of the first three enzymes were obtained by gene synthesis (Integrated DNA Technologies, IDT) after codon usage optimization for N. benthamiana. As for the fourth one (FAO) we tried to amplify it from the genomic DNA of the yeast Candida tropicalis, with no successful results. Nevertheless, the three synthetic genes were sufficient to produce at least two of our target pheromones, the alcohol (Z)-11-hexadecen-1-ol and the acetate (Z)-11-hexadecenyl acetate.

Figure 1. Engineered pheromone production pathway.

All three DNA sequences were domesticated; that is, standardized as GoldenBraid parts and cloned in the pUPD plasmid. Subsequently, each CDS was assembled with the strong constitutive Cauliflower mosaic virus promoter (P35S) and its terminator (T35S) respectively, in a multipartite assembly reaction. P35S is a strong plant constitutive promoter with high expression levels. As result of these assemblies, we obtained three Transcriptional units (TU) ready for plant transformation.

To maximize the flow through of the pathway, we wanted to make sure that all three genes were co-delivered simultaneously. This is to ensure that each transformed cell receives a complete set of genes and that the expression of all three enzymes is balanced and coordinated. Co-delivery is achieved by creating a multigenic construct, where all three genes are assembled in a single plasmid.

We used the GoldenBraid assembly system to create the multigene assembly. After two binary reactions, the 3-genes construct was obtained. This construct was then transformed, by agroinfiltration, into N. benthamiana plants for pheromone production.

We used GoldenBraid 2.0 for all our cloning reactions. GoldenBraid and BioBrick parts are not directly exchangeable; however, we adapted the coding sequences of the three biosynthetic genes to BioBrick standards using a GoldenBraid-Biobricks translator developed by the NRP-UEA-Norwich team. These BioBricks have been submitted to the Parts Registry.

RESULTS-CONSTRUCTS-PHEROMONE RELEASE

To obtain a plant capable of releasing pheromones into the environment in an efficient way, we decided to use the glandular trichome specific promoter (PCPS2) (see Pheromone Relase).

We obtained this promoter from Nicotiana tabacum genome and tested its functionality assembling it with GFP (see Results: Pheromone Release).

As it proved to be an effective promoter, expressing GFP only at the glandular cells of the trichomes, we decided to assemble each of the pheromone biosynthetic genes with this promoter to reach improvements in the pheromones release (see Biosynthesis).

Finally, after two binary GoldenBraid assembly steps, we obtained a multigenic construct with all three transcriptional units with the PCPS2 promoter. This construct was ready to be transferred to the plant to test the release of the pheromones.

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