Riggs C D, Voelker T A, Chrispeels M J
Department of Biology, University of California, San Diego, La Jolla 92093-0116.
Plant Cell. 1989 Jun;1(6):609-21. doi: 10.1105/tpc.1.6.609.
The effects of deleting DNA sequences upstream from the phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with respect to the level of gene product produced in the seeds of transgenic tobacco. Our studies indicate that several upstream regions quantitatively modulate expression. Between -1000 and -675, a negative regulatory element reduces expression approximately threefold relative to shorter deletion mutants that do not contain this region. Positive regulatory elements lie between -550 and -125 and, compared with constructs containing only 125 base pairs of upstream sequences (-125), the presence of these two regions can be correlated with a 25-fold and a 200-fold enhancement of phytohemagglutinin-L levels. These experiments were complemented by gel retardation assays, which demonstrated that two of the three regions bind cotyledon nuclear proteins from mid-mature seeds. One of the binding sites maps near a DNA sequence that is highly homologous to protein binding domains located upstream from the soybean seed lectin and Kunitz trypsin inhibitor genes. Competition experiments demonstrated that the upstream regions of a bean beta-phaseolin gene, the soybean seed lectin gene, and an oligonucleotide from the upstream region of the trypsin inhibitor gene can compete differentially for factor binding. We suggest that these legume genes may be regulated in part by evolutionarily conserved protein/DNA interactions.
就转基因烟草种子中产生的基因产物水平而言，已经研究了删除菜豆植物血凝素-L基因上游DNA序列的影响。我们的研究表明，几个上游区域在数量上调节表达。在-1000和-675之间，一个负调控元件相对于不包含该区域的较短缺失突变体，使表达降低约三倍。正调控元件位于-550和-125之间，与仅包含125个碱基对上游序列（-125）的构建体相比，这两个区域的存在可使植物血凝素-L水平分别提高25倍和200倍。凝胶阻滞分析对这些实验进行了补充，该分析表明，三个区域中的两个与中成熟种子的子叶核蛋白结合。其中一个结合位点位于一个与大豆种子凝集素和库尼茨胰蛋白酶抑制剂基因上游的蛋白质结合域高度同源的DNA序列附近。竞争实验表明，菜豆β-伴大豆球蛋白基因、大豆种子凝集素基因的上游区域以及胰蛋白酶抑制剂基因上游区域的一个寡核苷酸可以不同程度地竞争因子结合。我们认为，这些豆科植物基因可能部分受进化上保守的蛋白质/DNA相互作用调控。
