|Sheperd, Colin - ISU|
Submitted to: Biotech and Applied Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 16, 2008
Publication Date: January 29, 2009
Citation: Sheperd, C.T., Scott, M.P. 2009. Construction and Evaluation of a Maize Chimeric Promoter with Activity in Kernel Endosperm and Embryo. Biotech and Applied Biochemistry. 52:233-243. Interpretive Summary: Transgenic approaches can be used to improve the value of crops. One thing that limits transgenic technology is our ability to control gene expression so the product of a transgene is produced in the desired tissues and nowhere else in the plant. Using the protein GFP as a marker for transgene expression, we demonstrate that it is possible to develop a transgene with novel tissue specificity by fusing the promoters of two different genes into one chimeric gene. Our chimeric transgene is highly active in both embryo and endopsperm tissues of maize grain, but has minimal activity in other tissues of the plant. This promoter will allow production of high levels of beneficial foreign proteins in seeds while minimizing adverse effects associated with production of foreign proteins in tissues where they are not needed. This work will benefit the public by increasing our control over expression of transgenes, resulting in better products with reduced risk from the presence of transgene products in undesired tissues.
Technical Abstract: Chimeric promoters contain DNA sequences from different promoters. Chimeric promoters are developed to increase the level of recombinant protein expression, precisely control transgene activity, or to escape homology-based gene silencing. Sets of chimeric promoters, each containing different lengths of DNA from the maize 27kDa gamma zein (27zn) endosperm-preferred promoter and the Globulin-1 (Glb1) embryo-preferred promoter were created and tested in a transient expression assay of green fluorescent protein (GFP). Promoter fragments with the highest activity were combined to create the chimeric promoter A27znGlb1. In the context of the chimeric promoter, the selected Glb1 promoter fragment was necessary and sufficient to activate expression in embryo tissue and was functionally equivalent to the native Glb1 promoter. Similarly, the selected 27zn promoter fragment in the chimeric promoter was necessary and sufficient to activate expression in endosperm tissue and was functionally equivalent to the native 27zn promoter. Maize transgenic plants containing the A27znGlb1 chimeric promoter fused to GFP were produced to characterize this promoter in vivo. Quantitative reverse transcriptase PCR was used to determine that the promoter was active in the embryo, endosperm, pericarp, and immature leaf tissues. GFP activity in plants containing the chimeric promoter was not significantly different in endosperm than the activity of GFP fused to the full-length 27zn promoter, nor was it different in embryo than the activity of GFP fused to the full-length Glb1 promoter. Transgene copy numbers were shown to be between 4 and 12 copies in different events.