|Authors: ||L.A.I. Vaas, M. Marheine, E. Heine-Dobbernack, H.M. Schumacher, H. Kiesecker|
|Keywords: ||IRES efficiency, expression monitoring, Nicotiana benthamiana, vector architecture|
Dicistronic vector constructs harboring an internal ribosomal entry site (IRES) element facilitate the coordinated co-expression of physically independent proteins, as, e.g., a target protein along with a reporter protein.
Dicistronic vector constructs have been studied intensively in mammalian cell systems, but their efficiency and applicability in plant cell systems is much less known.
The influence of the 1st cistron nucleotide sequence on the translational expression level of the reporter gene encoded by the 2nd cistron was investigated.
The testing set consists of the plant viral TMV IRES element and the mammalian viral polio IRES element in combination with either the Atnhx gene, coding for a Na+/H+ antiporter, or the gus gene.
Using the resulting four combinations the impact on a firefly luciferase (luc) as reporter gene was studied at the transcriptional, translational and phenotypical level.
In order to discriminate between nucleotide sequence of the 1st cistron and its gene product, we introduced a frame shift in the 1st cistron coding sequence.
The sequence of the IRES element strongly influenced the translational reporter gene expression present in the 2nd cistron.
The results show that the dicistronic vector construct has to be carefully designed and tested for each individual application to obtain the desired level of translational expression of the reporter gene.
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