Molecular Cloning and Characterization of Nitrite Reductase Gene from Sugarbeet

Abstract

The nitrite reductase (NiR) gene was cloned using RT-PCR and 3′/5′RACE techniques. The cDNA of NiR gene isolated from sugarbeet was 2 014 bp containing a 1 830 bp opening-reading frame (ORF), encoding 599 amino acids. Further comparison showed that NiR gene had high homology to both of Spinacia oleracea NiR gene and Arabidopsis thaliana NiR gene, which was 93%.The predicted NiR protein found to have a hemoprotein beta-component (ferrodoxin-like), and 4Fe-4S region, its 3D structure was predicted by analysis software. Real time PCR analysis showed that when using 0, 10, 20, 30, 40, 50, 80, and 160 mmol L–1 NO3–-N treated for 72 hours, the expression of NiR gene was the highest at 50 mmol L–1;when using 0, 2, 4, 8, 16, 32, 64, and 128 mmol L–1 NH4+-N treated for 48 hours, the expression of NiR gene showed two peaks at 8 mmol L–1 and 64 mmol L–1, re-spectively. Furthermore, in experiments treated with different ratios of NO3–-N to NH4+-N for 48 h, the expression of NiR gene was the highest when the ratio was 80:20. Cycloheximide 9 h treatment experiments showed that NiR gene expression decreased with increasing the treating concentration. NO2–treatments indicated that the maximum expression of NiR gene was induced by 40 mmol L–1 NO2–.