Inaba T, Nagano Y, Sakakibara T, Sasaki Y
Laboratory of Plant Molecular Biology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Plant Physiol. 1999 Jun;120(2):491-500. doi: 10.1104/pp.120.2.491.
The pra2 gene encodes a pea (Pisum sativum) small GTPase belonging to the YPT/rab family, and its expression is down-regulated by light, mediated by phytochrome. We have isolated and characterized a genomic clone of this gene and constructed a fusion DNA of its 5'-upstream region in front of the gene for firefly luciferase. Using this construct in a transient assay, we determined a pra2 cis-regulatory region sufficient to direct the light down-regulation of the luciferase reporter gene. Both 5'- and internal deletion analyses revealed that the 93-bp sequence between -734 and -642 from the transcriptional start site was important for phytochrome down-regulation. Gain-of-function analysis showed that this 93-bp region could confer light down-regulation when fused to the cauliflower mosaic virus 35S promoter. Furthermore, linker-scanning analysis showed that a 12-bp sequence within the 93-bp region mediated phytochrome down-regulation. Gel-retardation analysis showed the presence of a nuclear factor that was specifically bound to the 12-bp sequence in vitro. These results indicate that this element is a cis-regulatory element involved in phytochrome down-regulated expression.
pra2基因编码一种属于YPT/rab家族的豌豆（Pisum sativum）小GTP酶，其表达受光敏色素介导，在光照下被下调。我们分离并鉴定了该基因的一个基因组克隆，并在萤火虫荧光素酶基因前构建了其5'上游区域的融合DNA。在瞬时分析中使用该构建体，我们确定了一个足以指导荧光素酶报告基因光照下调的pra2顺式调控区域。5'端缺失分析和内部缺失分析均表明，转录起始位点上游-734至-642之间的93 bp序列对光敏色素下调至关重要。功能获得分析表明，该93 bp区域与花椰菜花叶病毒35S启动子融合时可赋予光照下调功能。此外，接头扫描分析表明，93 bp区域内的一个12 bp序列介导了光敏色素下调。凝胶阻滞分析表明，体外存在一种与12 bp序列特异性结合的核因子。这些结果表明，该元件是一个参与光敏色素下调表达的顺式调控元件。
