RESUMO
Genes encoding beta-conglycinin, a soybean seed storage protein, are expressed only in seeds during mid-to-late stages in embryogeny. It was previously determined that a DNA sequence 200 nucleotides upstream of the transcriptional start site of the gene encoding the alpha'-subunit of beta-conglycinin is essential for regulated gene expression in transgenic plants. The regulatory effect of this DNA element was tested by inserting the element in different positions and different orientations within a chimeric constitutively expressed reporter gene. The reporter gene was comprised of the 35S promoter from cauliflower mosaic virus (CaMV), a gene encoding chloramphenicol acetyltransferase (CAT) and the polyadenylation signal from the alpha'-subunit gene. The element had no significant effect on the expression of the CAT gene in roots, stems, or leaves, regardless of the position of its insertion (i.e. 5 or 3 of the gene). However, there was 25- to 40-fold enhancement of CAT gene expression in seeds during mid-to-late stages of embryo development when the element was placed in either orientation within the 35S promoter. There was 2- to 4-fold enhancement of CAT activity when the element was placed 3' of the CAT coding sequence. No enhancement was detected when the element was placed downstream of the 3' non-coding region. This is, to our knowledge, the first identification of a cis-acting element that enhances gene expression in a tissue-specific and temporally regulated manner during embryo development in plants.
RESUMO
Soybean (Glycine max (L.) Merr.) seeds contain the storage protein ß-conglycinin, encoded by a multigene family. ß-Conglycinin consists of three subunits; α', α, and ß. A genomic clone for a ß-subunit of ß-conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this ß-subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The ß-subunit expressed in seeds of petunia and tobacco was recognized by anti-ß-conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the ß-subunit were produced. There was approximately a twofold variation in the accumulation of the ß-subunit protein in the mature seeds of transgenic petunia plants, each containing a single ß-subunit gene. However, the level of protein accumulation in mature seeds and the amount of ß-subunit mRNA in developing seeds was not correlated. Accumulation of the ß-subunit protein in transgenic seeds was less than the α'-subunit protein that accumulated in transgenic petunia seeds containing a single α'-subunit gene and less than the amount of the ß-subunit in mature soybean seeds which contain 8-13 ß-subunit genes. In transgenic tobacco plants, the accumulation of the ß-subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.