Molecular Cloning And Expression Of Nitrite Reductase Gene In Sugar Beet (beta Vulgaris L.)

Sugar beet (Beta vulgaris L.) was an endemic sugar crop and played an important role in agricultural production in north China.Its yield was secondary to sugarcane, and beet sugar was about 30% of the world's sugar yield.China was one of the big country which produced beet sugar and cane sugar.Planted area of sugarbeet in China was reached 67 million ha,which ranked 3rd in the world,and.total output was resides the 6st..But yields of sugar beet were not high and the total output was instable, particularly sugar content showed a downward trend in Heilongjiang province and the national. All had become the obstacle factors to the development of sugar beet. Unreasonable application of nitrogen was one of the main reasons besides the breeding lag .As nitrogen source and nitrogen metabolism played an central role in crop yield,determined the gene network of nitrogen regulation, identified the function of important genes, clarified the biochemical characteristics of gene expression protein, all will have a wide range influence on agricultural development.Nitrogen was the key and active factor in the necessary nutrients, and excess nitrogen can cause the reduction of sugar production in the sugar beet.Nitrate (NO3-) was the the main form of available nitrogen in dry crops, NR was the rate-limiting enzyme of NO3- inorganic assimilation,and NiR was the control enzyme of NO3- inorganic assimilation. Compared with the NR, few studies on NiR.Nitrite reductase (NiR) was a key enzyme of nitrogen assimilation pathway.NiR activity generally much higher than NR activity,so NiR as a limiting step of nitrate reduction was ruled out.Resulting in primary nitrogen assimilation studies neglected to its role of control enzyme at home and abroad in the past, and caused an unbalanced situation that emphasis on the NR in inorganic nitrogen assimilation metabolism.Modern molecular biotechnology methods was used in this study.NiR cDNA complete cds and NiR gDNA full length sequence of sugarbeet was first cloned; The peptide sequence and the functional domain of sugar beet NiR was identified for the first time; The 3D conformation of sugar beet NiR was conjectured for the first time;The expression of NiR gene on the transcription level was studied by RT-PCR under different proportion of NO3-N and NH4+-N, different NO3--N , different NH4+-N, then revealed the relationship of nitrogen and NiR gene expression in sugar beet ; Also on the basis of the nitrogen-induced, different concentrations of cycloheximide treatment , NO2- treatment and NaCl treatment was seted, which in order to study the NiR gene expression.The environmental factors on nitrite reductase gene expression was preliminary studied.The specific study contents and conclusions are as follows:1.According to the partial DNA sequence of beet (AF173663)and the mRNA sequence of Spinach(X07568)on GenBank , primers was designed in the conserved region, nitrite reductase gene 5′and 3′unknown region was amplified by RACE technique..2.Primers was designed according to the nitrite reductase gene 5′and 3′sequence , intermediate part was amplified.3.Taked the splicing sequences of 5′, 3′and P0 as the target sequences to design primers, NiR gene was firstly cloned from sugar beet. The accession number was HQ224499. The full length of NiR was 2014bp.The sequence included an ORF with 1830bp, encoded 599 amino acids,The cDNA and amino acid sequences showed high identity with other plant NiR genes by BLAST analysis.4.Sugar beet NiR genomic DNA was isolated by PCR and its sequence contained 4 exons and 3 introns. The accession number was HQ419065. The full length of genomic DNA was 2815bp, and the lengths of introns were respectively 531bp, 135bp and 269bp; the lengths of exons were respectively 437bp,355bp,289bp and 799bp.5.Physical and chemical properties of NiR was predicted with different methods,and the NiR phylogenetic tree was constructed.The results of primary structure analysis showed that NiR was soluble and hydrophobic, with a pI (isoelectric point) of 7.21 and MW (molecular weight) of 66.88kDa. A transmembrane region was found in C-end . The homology of spinach NiR and sugar beet NiR was the highest.Secondary structure analysis indicated that NiR contained 29.05% alpha helix, 21.04% extended strand and 49.92% random coil. Helix and coil structures were the main body of secondary structure .3D structure was constructed by homology method and contained 352 H-bonds,21 Helices,30 Strands,66 Turns.6.Protein structure was analysed, the amino acid sequence had a complete structure .The predicted NiR protein found to have a hemoprotein beta-component (ferrodoxin-like), and 4Fe-4S region, NiR was non-secreted protein with no signal peptide. The region TGCpnsCgqvqvaDIGF was the active site of NiR.7.Real time PCR analysis showed that, 0, 10, 20, 30, 40, 50, 80, and 160 mmol L–1 NO3–-N processing 72 hours experiments, the expression of NiR was higher at 50 mmol L–1; 0, 2, 4, 8, 16, 32, 64, and 128 mmol L–1 NH4+-N processing 48 hours experiments, the expressions of NiR were higher at 8 mmol L–1and 64 mmol L–1, respectively; Furthermore, in different nitrogen forms and ratios treatment 48 hours experiments, The expression of NiR gene was higher when the ratio of NO3–-N and NH4+-N was 80:20; Cycloheximide treatment nine hours experiments showed that NiR expression decreased with increasing treatment concentration; NO2– treatments indicated that the maximum expressions of NiR was induced by 40 mmol L–1NO2–; The expression of NiR had no significant law under different concentration NaCl treatment.