Isolation and molecular characterization of nitrite reductase (RsNiR) gene under nitrate treatments in radish
2015
Wu, Yue | Zhang, Wei | Xu, Liang | Wang, Yan | Zhu, Xianwen | Li, Chao | Liu, Liwang
Nitrite reductase (NiR, EC1.7.7.1) is a key enzyme that catalyzes the second step of nitrite reduction to ammonium in the process of nitrate assimilation in plants. To study the molecular mechanism underlying nitrate accumulation during plant development, the molecular characterization of the NiR gene (RsNiR) and its expression patterns under different nitrate concentrations and induction periods of nitrate treatments in leaf, root, petiole, stem and lateral root of radish (Raphanus sativus L.) were investigated in this study. Based on our radish transcriptome database, the genomic DNA sequence of RsNiR was isolated with four exons and three introns. The cDNA length was 1756bp containing an open reading frame (ORF) of 1524bp. The deduced protein consists of 506 amino acids, and showed highest identity with NiRs from Arabidopsis. Moreover, using the genomic DNA walking approach, the 843-bp 5’-flanking region upstream of RsNiR was isolated, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and nitrate-responsive cis-element (NRE). With the increasing nitrate concentration, the NR activity (NRA), nitrate contents and RsNiR expression levels exhibited increasing trends in leaf and root, reached a maximum at 30mM nitrate treatment and then decreased. In contrast, there was no obvious relationship among RsNiR expression level, NRA and nitrate content under different induction periods at 30mM nitrate. These results could provide fundamental insight into clarification of the molecular regulation mechanism underlying nitrate assimilation in radish.
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