Molecular Cloning Of Nitrite Reductase (Nii2) Gene And Its Expression In Tobacco

Nitrite Reductase (NiR) is an enzyme that can reduce the concentration of nitrite. Nitrite is one of the precursors of tobacco-specific nitrosamines (TSNA) which are potentially carcinogenic products and exist only in tobacco and tobacco products. In the second step of the nitrate assimilation pathway, ferredoxin-nitrite reductase (NiR) catalyzes the reduction of nitrite to ammonium, thus controlling this gene would influence the build-up of nitrite in nitrate assimilation pathway. In this study, we cloned the nii2gene from tobacco, and identified its function when it was overexpressed in tobacco. We further studied the relationship between nii2gene and TSNA, and investigated the NR and NiR activities, and their response to the excess amount of external nitrate and nitrite levels in tobacco. The detail results are summerised as follows:1. Cloning of the Ntni2gene and construction of the plant expression vector, pBI121-Ntnii2The Ntnii2gene was cloned from tobacco NC89based on the homologous information in Genbank (Accession number EST AB103507). The complete open reading frame (ORF) of Ntnii2gene contained1764bp in length andencoded587amino acids. The Ntnii2ORF was constructed into binary vector pBI121which has35S promoter of Cauliflower mosaic virus (CaMV), resulting in the pBI121-Ntnii2.2. Transformation and characterization of Ntnii2geneBy using Agrobacterium-mediated transformation method,23transgenic tobacco lines were generated through kanamycin resistance screening. After RT-PCR-based screening, NtNii2gene was confirmed to be successfully transcripted and inherited into its16offsprings while8lines were selected for further generation. Semi-quantitative RT-PCR results showed that the NiR gene expression in transgenic lines was significantly higher than that in WT plants.3. Study of physiological characters of transgenic plantsIn vivo and in vitro NR activities were significantly higher in those selected8transgenic lines than the counterpart WT plants. Approximately30%NiR activity was increased in some transgenic lines. When the nitrate content showed nearly46%reduction, the nitrite contents were not considerably differed from that of non-transformed plants. As a consequence among nitrate, nitrite and TSNA, our results can conclude that transgenic action would influence TSNA levels in tobacco.