Cloning of nia Gene and Differential Gene Expressions Induced by Nitrate and Ammonium Nitrogen in Sugar Beet (Beta vulgaris L.)

Abstract

Gene nia plays an important role in regulation of nitrogen metabolism in plants. This gene has been cloned in many crops except for sugar beet. In this study, a cDNA clone associated with nitrate reduction was isolated from a diploid species of sugar beet (Beta vulgaris L.) using homologous cloning technique. A novel full-length cDNA (GenBank accession number EU163265), termed nia, was obtained using rapid amplification of cDNA ends (RACE). The full length of nia gene is 3247 bp. The open reading frame of this gene is 2718 bp in length, which encodes 905 amino acids with the theoretical molecular weight of 102 kD and the isoelectric point of 6.12. Southern blotting results proved that nia presents in low copies in Ty7, and the clone obtained belongs to NADH-NR genes. The nia transcripts under nitrate nitrogen (NO₃-N) and ammonium nitrogen (NH₄-N) treatments were detected through semiquantitative PCR analysis, and the efficiency of mRNA synthesis from each sample was estimated via quantitative PCR of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the NH₄-N treatment, nia was up-regulated with low concentration of NH₄⁺, but inhibited by high concentration of NH₄⁺. In the NO₃-N treatment, the nia expression level was enhanced with the increase of NO₃⁽ concentration. When exposed to 30 mmol L⁽¹ KNO₃, nia exhibited the highest expression 4 h after treatment and a significantly reduction 6 h after treatment.