Kwon H B, Park S C, Peng H P, Goodman H M, Dewdney J, Shih M C
Department of Biological Sciences, University of Iowa, Iowa City 52242.
Plant Physiol. 1994 May;105(1):357-67. doi: 10.1104/pp.105.1.357.
We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.
我们在此报告了对拟南芥叶绿体甘油醛-3-磷酸脱氢酶B亚基编码核基因（GapB）光调控相关顺式作用区域的鉴定。我们的结果表明，一个664 bp的GapB启动子片段足以使转基因烟草（烟草）植株中大肠杆菌β-葡萄糖醛酸酶报告基因（Gus）实现光诱导和器官特异性表达。缺失分析表明，GapB基因上游-261至-173区域对于光诱导至关重要。该区域包含四个具有一致序列5'-ATGAA(A/G)A-3'（Gap框）的直接重复序列。所有四个重复序列的缺失完全消除了光诱导。此外，我们将一个包含四个直接重复序列的109 bp（-263至-152）GapB上游片段以两种方向与花椰菜花叶病毒35S基础启动子的-92至+6上游序列相连。所得的嵌合启动子能够赋予光诱导能力，并增强转基因烟草植株中Gus报告基因的叶特异性表达。基于这些结果，我们得出结论，Gap框对于拟南芥中GapB基因的光调控和器官特异性表达至关重要。通过凝胶迁移率变动分析，我们还鉴定出一种来自烟草的核因子，它与包含这些Gap框的GapA和GapB DNA片段相互作用。竞争分析表明，Gap框是该因子的结合位点。尽管这种结合活性存在于光照生长或暗处理烟草植株的叶和根的核提取物中，但在暗处理植株叶片或温室种植植株根制备的核提取物中活性较低。此外，我们的数据表明，这种结合因子与GT-1因子不同，GT-1因子与豌豆RbcS-3A基因光响应元件内的Box II和Box III结合。
