Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan.
Plant Cell Physiol. 2010 Jan;51(1):80-90. doi: 10.1093/pcp/pcp169. Epub 2009 Nov 24.
The molecular basis underlying the development, maintenance and function of companion cells in plants is largely unknown. The identification of several genes expressed specifically in companion cells implies the contribution of specific transcriptional elements to the identity of companion cells. However, less is known about the companion cell-specific transcriptional regulation of promoters. We established a novel assay method using gene-gun delivery of partially deleted promoters to string-containing vascular bundles excised from the petiole of Brassica napus for the rapid identification of cis-elements. To test this system, we analyzed the Arabidopsis METALLOTHIONEIN 2B (MT2B) gene, which is expressed in companion cells. The assay revealed a 49-bp region possessing two predicted cis-regulatory elements: a G-box and an evening element-related sequence (EEr), and EEr showed higher activity. We confirmed the reliability of the result with stable transformants harboring a deleted MT2B promoter:GUS transgene. The lack of EEr completely eliminated the MT2B-like expression, but the lack of G-box did not eliminate it. We conclude that EEr is a major cis-regulatory element of the MT2B promoter. Our method will help to explain the transcriptional background of companion cells through the rapid identification of cis-regulatory regions.
植物伴胞发育、维持和功能的分子基础在很大程度上是未知的。鉴定出几种在伴胞中特异性表达的基因，表明特定转录因子对伴胞的特性有一定的贡献。然而，关于启动子的伴胞特异性转录调控知之甚少。我们建立了一种新的测定方法，使用基因枪将部分缺失的启动子传递到从小白菜叶柄中分离出来的含串珠的维管束中，以快速鉴定顺式元件。为了测试该系统，我们分析了拟南芥金属硫蛋白 2B（MT2B）基因，该基因在伴胞中表达。该测定揭示了一个具有两个预测顺式调控元件的 49 个碱基对区域：一个 G 框和一个晚元件相关序列（EEr），并且 EEr 表现出更高的活性。我们通过含有缺失的 MT2B 启动子：GUS 转基因的稳定转化体证实了结果的可靠性：GUS 转基因。缺乏 EEr 完全消除了 MT2B 样表达，但缺乏 G 框并没有消除它。我们得出结论，EEr 是 MT2B 启动子的主要顺式调控元件。我们的方法将通过快速鉴定顺式调控区来帮助解释伴胞的转录背景。
