Difference between revisions of "Part:BBa K3890000"

Latest revision as of 19:52, 21 October 2021

Pollen expressed CYP6G1 and GUS reporter circuit

Circuit that allows CYP6G1 to be expressed specifically in pollen tissue to metabolize pesticides locally. The enzyme and the GUS reporter are expressed under the same promoter, pLAT52, because of the self cleaving LP4/2A peptide between them.

Usage and Biology

The circuit was designed by USP-Brazil team for a specific pollen expression of a metabolizing pesticide enzyme. That allows both the pollen to be safer for bees’ pollination and the plant to stay safe against pests by keeping the function of the substance in other regions of its body. The chosen enzyme was the CYP6G1, from Drosophila melanogaster, codon optimized for tomato expression, proven to metabolize imidacloprid to less harmful metabolites: 4-hydroxy and 5-hydroxy []. It has a LP4/2A fusion cleaving peptide for efficient co-expression of a second protein with the CYP6G1 metabolizing enzyme, the GUS reporter needed for lab experiments to confirm the promoter expression [2]. The use of the peptide has lowered the cost and the complexity of the circuit, allowing the use of only one copy of the promoter.

Characterization

During the development of the project, in 2020 and 2021, the COVID-19 pandemic limited the team’s access to the labs, hindering the performance of some experiments. Nonetheless, it was possible to gather modelling results and preliminary results done in the plant’s T0 generation concluding the efficiency of the pollen specific expression and metabolization efficiency, that can be complemented in the future.

RT-qPCR was performed to detect our circuit's transcript and compare with other plants that do not have it for prove our functionality construction. Significative expression at transcript level for CYP2 (median Ct value of 21.4) and GUS (median Ct value of 20.5) genes were detected only for pollen of transgenic plants (median Ct values were above 31.9 for both genes in cDNA samples from pollen and leaves of control plants and from leaves of transgenic plants, as well as undetermined for both genes in negative control). These findings confirm the specific expression of CYP6G1 and GUS genes, at least at the transcriptional level, in the pollen of transgenic tomato plants and its functionality to produce in vivo transcripts (Table 1).

Table 1. Median Ct values from duplicates from RT-qPCR for measure CYP6G1, GUS and Expressed transcripts from leafs and pollen.

Finally, a GUS histochemical assay was also carried out, by detection of the characteristic blue coloring. Micro-tom 2 and 22 were confirmed to presence of GUS activiy. As we could not get pollen from plant 2 genomic PCR confirmed the presence of the construct in both lineages. Even though it was not possible to extract fresh pollen from plant number 2, pollen grains attached to its ovarium walls were stained during the flower staining experiment. Nevertheless, pLAT52-CYP6G1_LP4/2A_GUS expression was later confirmed in plant number 2 through RT-qPCR.

Figure 1. Presence of GUS activity on plants. (-C) - Pollen negative control. (+C) - Leaf positive control (GUS expressed by CaMV35S promoter). 2 and 22 - Transformants Micro-tom for our circuit. Blue pollen cells are indicated by arrows in pictures 2 and 22.

Through mathematical modelling and simulations, we predict that the total metabolization of imidacloprid occurs in 12 seconds. It may look unrealistic at a first glance, but one should bear in mind that this model attains to a single compartment - the pollen. We also found out that, when simulating a scenario with beehives around plants transformed with Let.it.bee., in comparison to wild plants, plants transformed with Let.it.bee. can be 5 to 10 times less toxic than wild type plants. In some cases, our project can avoid the extinction of a whole colony.

References

JOUßEN, N., Heckel, D. G., Haas, M., Schuphan, I., & Schmidt, B. (2007). Metabolism of imidacloprid and DDT by P450 CYP6G1 expressed in cell cultures of Nicotiana tabacum suggests detoxification of these insecticides inCyp6g1-overexpressing strains ofDrosophila melanogaster, leading to resistance. Pest Management Science, 64(1), 65–73. doi:10.1002/ps.1472

SUN H, Zhou N, Wang H, Huang D, Lang Z. Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system. PLoS One. 2017 Mar 30;12(3):e0174804. doi: 10.1371/journal.pone.0174804. PMID: 28358924; PMCID: PMC5373624.

Sequence and Features