Iowa State University
Pioneer Hi-Bred International, Inc.

Structural and functional elements of the 5' region of the P-rr gene
--Xianggan Li, Laura Tagliani, Lyudmila Sidorenko, Ben Bowen, and Thomas Peterson

The P-rr gene conditions the synthesis of a phlobaphene-like red pigment in mature cob glumes, pericarps, and husks. The P-rr gene is expressed at a relatively low level, predominantly in the female inflorescence, and at a late stage of development. These characteristics suggest that the P-rr promoter could be useful for directing the expression of foreign genes for pathogen resistance specifically in pericarps, silks, and cob glumes. The P-rr 5' region contains a Tourist-like mobile element located approximately 1 kbp 5' of the transcription start site; the same element is found at the identical site in the 5.2 kbp direct repeat 3' of the P-rr gene, suggesting that insertion of the Tourist-like element occurred prior to duplication of the 5.2 kbp direct repeats flanking P-rr.

Functional analysis of the P-rr promoter via transient assay gave the following results. A basal construct, containing the GUS reporter gene, Adh1 intron, 220 bp P-rr untranslated leader and 240 bp upstream from the P-rr transcription start site, gives a low but detectable level of GUS expression when introduced via microprojectile bombardment into pericarps. Addition of the adjacent 5' 1.0 kb P-rr fragment containing a tRNA-homologous sequence, or a 1.2 kb SalI fragment located 4.6 kb 5' of the P-rr transcription start site, boosts the activity of the basal construct about 10-fold. The increased expression suggests that both fragments from the P-rr 5' region contain enhancer elements. Because Ac insertions in either fragment can reduce P-rr expression in vivo (Moreno et al., Genetics 131:939-956), both sequences may be important for P-rr expression. The extended P-rr promoter construct containing the 1.24 kbp immediately upstream of the 5' start site directs the expression of the GUS reporter gene preferentially in pericarp compared to scutellum when the amount of DNA used is lowered to 100 pg per bombardment. We have initiated stable transformation experiments with these constructs to analyze the activity and tissue specificity of the P-rr promoter in transgenic maize plants. 

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