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To study the regulatory expression mechanism of soybean glycinin gene, Gy2, the 5´ upstream region of the gene was searched for the presence of putative regulatory elements by nucleotide sequencing. It revealed various kinds of regulatory sequence elements commonly found in plant storage protein genes. There were canonical promoter sequences, TATA box (TATAAT) and AGGA box (GAAT) which are common in the 5´ upstream region of the plant genes. The embryo factor binding sequence, RY repeat, CACA sequences, α-conglycinin enhancer-like sequences were also found. To delineate the function of these sequences, 5´ upstream deletion mutants of Gy2 were prepared and fused to the α-glucuronidase (GUS) gene. Each chimeric construct was transferred into soybean protoplasts for transient assay, which led to the identification of the sequences between -281 and - 223, -170 and -122, of Gy2 promoter as negative regulatory elements, and the sequences between -223 and -170, -122 and -16 as positive regulatory elements. These results are consistent in transformed tobacco plants as well. The serially deleted promoter fragments fused to the GUS were transformed into Nicotiana tabacum by Agrobacterium tumefaciens using the binary vector system. GUS activity of Gy2 promoter deletion constructs was detected only in seeds but not in leaves with different levels of expression as in transient assay. These results suggest that the glycinin Gy2 promoter drives a tissue-specific expression in transgenic tobacco plants.
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