Functional analysis of regulatory elements in a plant embryo-specific gene.

Previously we demonstrated the expression of a plant embryo-specific gene encoding the alpha' subunit of beta-conglycinin, a seed storage protein of soybean (Glycine max), in transgenic petunia plants. To examine the regulatory elements that control the expression of this embryo-specific gene (Gmg17.1), a series of deletion mutants was made that contain the alpha'-subunit gene flanked in the 5' direction from +14 nucleotides to -8.5 kilobases (kb) relative to the site of transcription initiation. Each of these deletion mutants was introduced into the genome of petunia cells with the help of Ti-plasmid-derived vectors. Petunia plants were regenerated from transformed cells and expression of the introduced soybean gene was examined. When the alpha'-subunit gene was flanked by 159 nucleotides upstream (Gmg17.1 delta-159), the gene was expressed at a low level in immature embryos. When the gene was flanked by 257 nucleotides upstream of the site of transcription initiation (Gmg17.1 delta-257), a high level of expression was obtained. An additional 8 kb of DNA sequence (which includes the sequence GTGGATAG at -560, which is identical to the core enhancer sequence of simian virus 40 and some animal genes) did not significantly increase the level of expression. The increase in expression level between the delta-159 and delta-257 mutants was at least 20-fold. Analysis of the nucleotides between delta-159 and delta-257 reveals four repeats of a 6-base-pair (G + C)-rich sequence (see formula in text). The deletion Gmg17.1 delta-159 contains a single AACCCA sequence. We suggest that the (G + C)-rich repeats play a critical role in determining the level of expression of the transgenic plants.