Identification of a novel Cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5′-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber
Identification of a novel Cis-element exhibiting cytokinin-dependent protein binding in vitro in the 5′-region of NADPH-protochlorophyllide oxidoreductase gene in cucumber
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DOI：
10.1007/s11103-005-0579-x
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发表时间：
2005-11-01
影响因子：
5.1
通讯作者：
Takamiya, K
中科院分区：
文献类型：
--
作者：
Fusada, N;Masuda, T;Takamiya, K
细胞分裂素和光激活黄瓜 NADPH-原叶绿素还原酶 (POR) 基因的转录。我们之前报道过该基因 5' 区的 2.3 kb 含有一个对细胞分裂素有反应的顺式元件。在本研究中，为了鉴定与叶绿素生物合成和叶绿体发育相对应的细胞分裂素响应顺式元件，我们在黄化的黄瓜子叶中进行了瞬时表达测定。 5'-缺失分析表明，411-bp 片段（-451 至-40 bp）包含至少一个与细胞分裂素响应性相关的顺式元件。凝胶迁移率变化测定还检测到该区域中细胞分裂素增强的结合。使用 150 bp 片段（-490 至 -340 bp）作为探针进行 DNase I 足迹分析，将细胞分裂素增强的保护序列鉴定为 5'-ATATTAGTGATAT-3'。更详细的凝胶迁移率变化和竞争分析确定 5'-TATTAG-3' 是细胞分裂素增强结合的关键序列。瞬时表达测定中鉴定的序列中的突变导致细胞分裂素反应性降低但保留，以及未处理对照的低报告活性。这些结果表明，所鉴定的序列是一种新的顺式元件，在体外表现出细胞分裂素依赖性蛋白质结合，当与其他细胞分裂素相关元件相互作用时，它可能有效地发挥作用。该元素对叶绿体发育的影响将与其他细胞分裂素相关元素一起讨论。
Cytokinins and light activate the transcription of the cucumber NADPH-protochlorophyllide reductase (POR) gene. We have previously reported that 2.3 kb of the 5'-region of this gene contains a cis-element that is responsive to cytokinin. In this study, to identify the cytokinin-responsive cis-element corresponding to chlorophyll biosynthesis and chloroplast development, we performed transient expression assays in etiolated cucumber cotyledons. A 5'-deletional analysis indicated that a 411-bp fragment (-451 to -40 bp) contained at least one of the cis-elements related to cytokinin-responsiveness. Gel mobility shift assays also detected cytokinin-enhanced binding in this region. DNase I footprinting analysis, using a 150-bp fragment (-490 to -340 bp) as the probe, identified the cytokinin-enhanced protected sequence as 5'-ATATTAGTGATAT-3'. More detailed gel mobility shift and competition analyses identified 5'-TATTAG-3' as the sequence critical for cytokinin-enhanced binding. Mutations in the identified sequence in the transient expression assay caused a reduced but retained cytokinin-responsiveness, as well as low reporter activity of untreated control. These results suggest that the identified sequence is a novel cis-element exhibiting cytokinin-dependent protein binding in vitro, which may function effectively when interacting with other cytokinin-related elements. The effects of this element on the chloroplast development are discussed in relation to other cytokinin-related elements.