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Développement d'un construct de gène rapporteur pour la localisation subcellulaire
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ISBN10: 6630014634
ISBN13: 9786630014631
Publisher: Editions Notre Savoir
Published: Jun 1 2026
Pages: 92
Weight: 0.30
Height: 0.22 Width: 6.00 Depth: 9.00
Language: French
ISBN13: 9786630014631
Publisher: Editions Notre Savoir
Published: Jun 1 2026
Pages: 92
Weight: 0.30
Height: 0.22 Width: 6.00 Depth: 9.00
Language: French
La pyrophosphohydrolase d'ARN (RppH) déclenche la dégradation des transcrits en éliminant le pyrophosphate de l'extrémité 5' des ARNm, ce qui permet la liaison de la RNase E/RNase J chez les bactéries. Une ARN pyrophosphorylase putative est présente dans les cellules de C. reinhardtii et plusieurs indices suggèrent que cette protéine est impliquée dans la dégradation de l'ARNm au sein du chloroplaste. Afin de déterminer la localisation de la protéine dans les cellules de Chlamydomonas, l'extrémité 5' du gène rppH a été marquée par une protéine fluorescente verte à codons optimisés et introduite dans des cellules de Chlamydomonas. La GFP (Zsgreen 1) a été optimisée au niveau des codons à l'aide d'une base de données sur l'utilisation des codons. Le cadre de lecture de la GFP synthétique optimisée a été ajusté et cloné dans le vecteur pBluescript-5'rppH SK+. Le fragment de gène 5'rppH-GFP a ensuite été cloné dans trois vecteurs. Toutes les constructions ont été vérifiées. Les transformants ont été criblés par PCR pour détecter la présence du gène GFP chimérique. Les expériences de criblage nous ont amenés à choisir un transformant pouvant être utilisé pour la suite des travaux. De plus, bien que la fluorescence n'ait pas été vérifiée, la GFP synthétique optimisée a été exprimée dans des cellules E. coli (BL21DE3), confirmant la fonctionnalité de la construction GFP synthétique in vivo.
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