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

Abstract

Four non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivars, Suzhouqing, Xuekeqing, Huangxinwu, Aijiaohuang, were planted to investigate the activity of nitrate reductase (NRA) in leaves. After being induced by KNO₃ at 50 mmol/L for different hours, the NRAs of the four cultivars were determined in vivo. The results showed that the NRAs changing trends of these four cultivars were similar. The highest NRAs in leaves reached their maximum at the 4th, 4th, 6th, and the 6th hour of induction, respectively. According to these results, the level of NR mRNA in plants could be enhanced by nitrate inducement. Then, the total RNA was isolated from the leaves of Suzhouqing that was induced by KNO₃ at 50 mmol/L for four hours, and two fragments of NR cDNA were obtained through RT-PCR using specific primers. The products of PCR were cloned and sequenced. They are 1 125 and 438 base pairs, which were named nr₁₁₂₅ and nr₄₃₈, encoding 374 and 135 amino acids, respectively. Finally, nr1125 was accepted and released by GenBank (accession number DQ001901).

Keywords

non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino), nitrate reductase, PCR, clone