Molecular cloning and characterization of nitrate reductase gene from non-heading Chinese cabbage

Nitrate reductase (NR) is a key enzyme that catalyzes the first step in plants nitrogen metabolization. A cDNA of a NR gene from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar ‘Suzhouqing’ was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3049 bp contained an open reading frame of 2733 bp encoding 910 amino acids, a 5′-untranslated region of 113 bp and a 3′-untranslated sequence of 203 bp with a poly (A) tail. This protein shares common structural features with NRs from other higher plants and eukaryotes. It was classified as NR by sequence alignment, motif search and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and consequently nominated Brassica campestris ssp. chinensis Makino NR (BcNR). Southern blot analysis indicated that BcNR gene represented as 3 copies in the non-heading Chinese cabbage genome. Accumulation of BcNR mRNA was transiently induced by nitrate supply and correlated with nitrate concentration, the highest mRNA levels of BcNR was induced by 30 mM NO₃⁻ and reached a maximum at 4 h at this optimal concentration treatment, in contrast, NH₄⁺ cannot induce the accumulation of BcNR mRNA. The BcNR was heterologously expressed in Escherichia coli BL21 (DE3) as a fusion protein. There was a specific band at about 110 kDa in size by SDS-PAGE analysis which was according with the expected molecular weight of the recombinant protein. To further explore its biological function, BcNR under regulation of the cauliflower mosaic virus (CaMV) 35S promoter was transferred into Arabidopsis thaliana. The transgenic plants exhibited an enhanced level of NR and nitrate reductase activity (NRA) in leaves under NO₃⁻ inducement.