Regulated expression of genes encoding soybean β‐conglycinins in transgenic plants

Abstract

β-conglycinin, an abundant storage protein in soybean seeds, is comprised of three subunits: α', α, and β. Expression of genes encoding these subunits is tissue-specific and temporally regulated in soybean plants. Both the α'-and β-subunit genes have been isolated and fully or partially characterized by DNA sequence analysis. Each has been transferred to transgenic plants and was found to be expressed in petunia and tobacco plants in a regulated manner as in soybean plants. Both the α'- and β-subunit proteins were accumulated only in transgenic seeds during mid-to-late stages of seed development and assembled into multimeric forms with sedimentation coefficients of 7S and 9S. The α'- and β-subunit genes were also introduced on the same plasmid into transgenic plants. When the promoters of the two genes were proximal to each other, there was an increase in the expression of the β-subunit gene, suggesting that there is a strong cis-acting sequence in the α'-gene (promoter) that can affect expression of the α-subunit gene. A series of deletion mutants was constructed in the α'-gene promoter, and each was introduced into petunia plants. The DNA sequence approximately 250 bp upstream of the transcriptional initiation site contains sufficient regulatory information for expression of the α'-subunit gene. The sequence about 200 bp upstream contains four 6-bp repeats A (AGCCCA) that may play an important role in regulating expression of the α'-gene. The function of this DNA sequence (-78 to -257) was tested in a chimeric gene construct containing the CaMV 35S promoter, the CAT coding sequence, and the 3′ noncoding region of the α'-gene. A dramatic enhancement of the CAT gene expression (at least 40-fold) resulted when this DNA sequence was inserted into the 35S promoter at a position 90 bp upstream of the transcription start site, regardless of the orientation of the insert. There was a two- to threefold enhancement when the element was placed 3′ to the CAT coding region and no enhancement when placed downstream of the 3′ noncoding region. The enhancement was observed only in seeds during mid-to-late stages of seed development. This indicates that the 170-bp DNA sequence can function as a strong cis-acting element that controls seed-specific and temporally regulated gene expression. This element is defined as a seed-specific and developmentally regulated element (SDRE).