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Entwicklung eines Reportergen-Konstrukts zur Bestimmung der subzellulären Lokalisierung
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ISBN10: 6630009517
ISBN13: 9786630009514
Publisher: Verlag Unser Wissen
Published: May 31 2026
Pages: 92
Weight: 0.30
Height: 0.22 Width: 6.00 Depth: 9.00
Language: German
ISBN13: 9786630009514
Publisher: Verlag Unser Wissen
Published: May 31 2026
Pages: 92
Weight: 0.30
Height: 0.22 Width: 6.00 Depth: 9.00
Language: German
Die RNA-Pyrophosphohydrolase (RppH) leitet den Abbau von Transkripten ein, indem sie Pyrophosphat vom 5'-Ende der mRNAs entfernt, was die Bindung von RNase E/RNase J in Bakterien ermöglicht. In Zellen von C. reinhardtii ist eine mutma liche RNA-Pyrophosphorylase vorhanden, und mehrere Hinweise deuten darauf hin, dass das Protein am mRNA-Abbau im Chloroplasten beteiligt ist. Um den Ort des Proteins in Chlamydomonas-Zellen zu bestimmen, wurde das 5'-Ende des rppH-Gens an ein codonoptimiertes grün fluoreszierendes Protein (GFP) gekoppelt und in Chlamydomonas-Zellen eingeführt. GFP (Zsgreen 1) wurde unter Verwendung einer Codon-Usage-Datenbank codonoptimiert. Der Leserahmen des optimierten synthetischen GFP wurde angepasst und in den Vektor pBluescript-5'rppH SK+ kloniert. Das 5'rppH-GFP-Genfragment wurde anschlie end in drei Vektoren kloniert. Alle Konstrukte wurden verifiziert. Die Transformanten wurden mittels PCR auf das Vorhandensein des chimären GFP-Gens untersucht. Die Screening-Experimente führten dazu, dass wir einen Transformanten auswählten, der für die weitere Arbeit verwendet werden konnte. Obwohl die Fluoreszenz nicht überprüft wurde, wurde das optimierte synthetische GFP in E. coli (BL21DE3)-Zellen exprimiert, was die Funktionalität des synthetischen GFP-Konstrukts in vivo bestätigte.
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