Jin G, Davey M C, Ertl J R, Chen R, Yu Z T, Daniel S G, Becker W M, Chen C M
Biomedical Research Institute, University of Wisconsin-Parkside, Kenosha 53141, USA.
Plant Mol Biol. 1998 Nov;38(5):713-23. doi: 10.1023/a:1006034932322.
Transcription of the cucumber hpr-A gene is responsive to cytokinin and light. To investigate the molecular basis for transcriptional regulation by cytokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (-382 to -67) contains sequences necessary for cytokinin responsiveness of the luciferase reporter gene. Band shift assays detected cytokinin-enhanced and -reduced protein binding sites in a 97 bp fragment (-382 to -285) upstream of the hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5'-AAATGACGAAAATGC-3', that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5'-AAGATTGATTGAG-3', of unknown function. Two-dimensional band shift analysis of the cytokinin-responsive DNA protein complex revealed the presence of six DNA protein interactions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share identical signal transduction pathways in regulating hpr-A gene expression.
黄瓜hpr - A基因的转录对细胞分裂素和光有响应。为了研究细胞分裂素转录调控的分子基础，我们鉴定了可能参与hpr - A基因表达调控的DNA序列和蛋白质。在黄化黄瓜子叶中的瞬时表达分析表明，315 bp片段（- 382至- 67）包含荧光素酶报告基因对细胞分裂素响应所必需的序列。凝胶迁移实验在hpr - A基因上游97 bp片段（- 382至- 285）中检测到细胞分裂素增强和减弱的蛋白质结合位点。DNase I足迹实验鉴定出两个蛋白质保护位点，一个15 bp序列5'-AAATGACGAAAATGC-3'，其包含在其他植物启动子中发现的as - 1 TGACG基序，以及一个功能未知的13 bp序列5'-AAGATTGATTGAG-3'。对细胞分裂素响应性DNA -蛋白质复合物的二维凝胶迁移分析揭示了六种DNA -蛋白质相互作用的存在。凝胶迁移实验表明，细胞分裂素和光对核蛋白与hpr - A基因97 bp片段的相互作用有不同影响。这些数据表明，在调控hpr - A基因表达方面，细胞分裂素和光不共享相同的信号转导途径。
