Rastogi R, Bate N J, Sivasankar S, Rothstein S J
Department of Molecular Biology & Genetics, University of Guelph, Ontario, Canada.
Plant Mol Biol. 1997 Jun;34(3):465-76. doi: 10.1023/a:1005842812321.
Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between -330 to - 200, was shown to be necessary for nitrate induction of beta-glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5' deletions in the -330 to -200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to -230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between -230 to -200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the -300 to -130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the -230 to -181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The -240 to -110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.
亚硝酸还原酶（NiR）是硝酸盐同化途径中的第二种酶，可将亚硝酸盐还原为铵。NiR基因的表达在添加硝酸盐后被诱导。在早期的一项研究中，菠菜NiR基因启动子的130 bp上游区域（位于-330至-200之间）被证明是转基因烟草植株中硝酸盐以组织特异性方式诱导β-葡萄糖醛酸酶（GUS）表达所必需的[28]。为了进一步确定参与NiR基因表达硝酸盐调控的顺式作用元件，构建了菠菜NiR基因启动子-330至-200区域5'端缺失并与GUS基因融合的转基因烟草植株。将NiR启动子缺失至-230的植株在硝酸盐存在下GUS活性显著增加，表明-230至-200之间的30 bp区域对NiR的硝酸盐调控表达至关重要。对两个独立转基因株系叶片组织中NiR启动子-300至-130区域进行体内二甲基亚砜足迹分析，发现了几个硝酸盐诱导的足迹。在-230至-181区域内的足迹分析显示，有因子结合到两个由24 bp隔开的相邻GATA元件上。这种GATA元件的排列类似于粗糙脉孢菌硝酸盐诱导基因启动子中发现的顺式调控序列，这些基因受NIT2锌指蛋白调控。NiR启动子的-240至-110片段包含两个NIT2共有核心元件，在体外与包含粗糙脉孢菌NIT2蛋白锌指结构域的融合蛋白结合。本文提供的数据表明，NiR基因的硝酸盐诱导表达是由反式作用因子与真菌和高等植物之间保守序列的硝酸盐特异性结合介导的。
