Isolation and molecular characterization of nitrite reductase (RsNiR) gene under nitrate treatments in radish

• The gDNA of RsNiR was firstly isolated according to the transcriptome database.
• The 5’-flanking region of RsNiR was firstly isolated with genome walking method.
• RsNiR expression was different in radish issues and highest in leaves and roots.
• A similar regulatory pattern of NRA and NiR mRNA was found in radish.
• Nitrate highly regulated the expression level of RsNiR, nitrate content and NRA.

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 1756 bp containing an open reading frame (ORF) of 1524 bp. 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 30 mM 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 30 mM nitrate. These results could provide fundamental insight into clarification of the molecular regulation mechanism underlying nitrate assimilation in radish.