Evaluation of factors which stabilize transgene expression in plants

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Regulation of gene expression in eukaryotes is a complex phenomenon. Over-expression of a gene may result in its “silencing”. This phenomenon called RNA silencing is an anti-viral defense mechanism in plants. Viruses encode certain proteins called suppressors of silencing, which suppress the RNA silencing defense mechanisms of plants. For our research, different plant viral suppressors were evaluated for their ability to stabilize expression of the Green Fluorescent Protein (GFP) using a unique transient expression assay. DNA was introduced into lima bean cotyledons via particle bombardment and automated image collection and analysis were used for quantification and tracking of GFP expression. Introduction of the gfp gene under regulatory control of the constitutive 35S promoter resulted in GFP expression lasting only for about 72 hrs post bombardment. However, introduction of DNAs with the gfp gene fused to the different viral suppressors resulted in significant extensions of GFP expression. HC-Pro (Tobacco Etch Virus) and P19 (Tomato Bushy Stunt Virus) were among the strongest stabilizers of transgene expression, extending GFP expression up to 120 hrs. Although it is still unclear if these viral suppressors are indeed functioning as silencing suppressors in this system, there is a clear extension and stabilization of transgene expression. Evaluation of the stabilizing effects of these DNA fragments using the lima bean cotyledon model, together with automated image collection and analysis, has provided a consistent mechanism for comparing the relative “strength” of the gene expression stabilizers. These stabilizers could potentially provide more consistent transgene expression in plants and other organisms.



Transgene expression stabilization, Suppressors of silencing