Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
Plant Cell Physiol. 2011 May;52(5):824-36. doi: 10.1093/pcp/pcr033. Epub 2011 Mar 30.
Nitrate reductase (NR) is the enzyme that catalyzes the first step of nitrate assimilation. It is well known that the expression of NR genes is rapidly induced in various plants by nitrate. Previously, the activity of a tobacco NR gene promoter was reported to be high in tobacco plants grown on medium containing ammonium as the sole nitrogen source, but low in tobacco plants grown on nitrate-containing medium. This cast some doubt on the role of the NR gene promoter in the nitrate-inducible expression of this gene. Furthermore, in previous studies, transformation with genomic fragments containing NR loci restored the reduced NR activity in NR mutants to a limited extent, suggesting a complex regulation of NR gene expression. Here, we show that although the 1.9 kb promoter of an NR gene in Arabidopsis, NIA1, is not activated by nitrate, the expression of a GUS (β-glucuronidase) reporter gene inserted between the 5'- and 3'-flanking sequences of the NIA1 coding region is strongly induced by nitrate. When the 3'-flanking sequence was fused downstream of the GUS gene under the control of the 35S minimal promoter, its expression was also strongly induced by nitrate. Furthermore, dissection analysis of the 3'-flanking region revealed that the sequence downstream of the transcriptional terminator rather than the 3'-untranslated region plays a role in nitrate-inducible expression, indicating a requirement for the 3'-flanking sequence for the nitrate-inducible transcription of NIA1. We also show that the 2.7 kb promoter sequence of NIA2, another NR gene of Arabidopsis, cannot direct nitrate-inducible expression.
硝酸还原酶（NR）是催化硝酸盐同化第一步的酶。众所周知，硝酸盐会迅速诱导各种植物中 NR 基因的表达。以前，据报道，在以铵盐作为唯一氮源的培养基中生长的烟草植物中，烟草 NR 基因启动子的活性很高，但在含有硝酸盐的培养基中生长的烟草植物中活性较低。这使得人们对 NR 基因启动子在该基因的硝酸盐诱导表达中的作用产生了一些怀疑。此外，在以前的研究中，用含有 NR 基因座的基因组片段转化可在一定程度上恢复 NR 突变体中还原型 NR 活性，这表明 NR 基因表达受到复杂的调控。在这里，我们表明，尽管拟南芥 NR 基因 NIA1 的 1.9kb 启动子不受硝酸盐激活，但插入 NIA1 编码区 5'和 3'侧翼序列之间的 GUS（β-葡萄糖醛酸酶）报告基因的表达被硝酸盐强烈诱导。当 3'侧翼序列在 35S 最小启动子的控制下下游融合到 GUS 基因后，其表达也被硝酸盐强烈诱导。此外，对 3'侧翼区的分析表明，转录终止子下游的序列而不是 3'非翻译区在硝酸盐诱导表达中起作用，表明需要 3'侧翼序列来进行 NIA1 的硝酸盐诱导转录。我们还表明，拟南芥另一个 NR 基因 NIA2 的 2.7kb 启动子序列不能直接指导硝酸盐诱导的表达。
