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Développement d’outils moléculaires et cellulaires pour générer des variétés de Pomelo « Star Ruby » ne produisant pas de Furocoumarines

Abstract : Furanocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants.
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Submitted on : Tuesday, June 8, 2021 - 2:53:01 PM
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  • HAL Id : tel-02347896, version 1



Mariana Cecilia Limones Méndez. Développement d’outils moléculaires et cellulaires pour générer des variétés de Pomelo « Star Ruby » ne produisant pas de Furocoumarines. Agronomie. Université de Lorraine, 2019. Français. ⟨NNT : 2019LORR0045⟩. ⟨tel-02347896⟩



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