Gene Cloning And Bioinformatics Analysis Of Nitrite Reductase 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. 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. Nitrite reductase(NiR) was a important enzyme in nitrogen metabolism.It couple with nitrate reductase(NR) in the assimilatory reduction of NO3- . At present, compared with NR,the researches of nitrite reductase structure and characteristics were reported few. In this study, SbNiR gene was cloned by degenerate PCR and 5'/3'-RACE from diploid sugar beet variety (Tianyan 7) and its structure and function was analyzed by bioinformatics. The regulation mechanism of sugar beet nitrite reductase could be revealed from the enzyme molecular structure. The results were as follows:1 SbNiR gene was firstly cloned from sugar beet by degenerate PCR and 5'/3'-RACE. The accession number was HQ224499. The full length of SbNiR was 2014bp and encoded 599 amino acids. The cDNA and amino acid sequences showed high identity with other plant NR genes. 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 532bp, 136bp and 270bp.2 Protein structure was predicted by different methods. The results of primary structure analysis showed that SbNiR was soluble and hydrophobic, with a pI (isoelectric point) of 7.21 and MW (molecular weight) of 66.88kDa. Secondary structure analysis indicated that SbNiR belonged to mixed protein class. The alpha helix,extended strand and Random coil of SbNiR were about 39%,15% and 48%.3D structure was constructed by homology method Geno3d. SbNiR have 24 alpha helix and 57 beta turns.3 nitrite reductase of the phylogenetic tree analysisi showed that SbNiR and chenopodiaxeae NiR closer relationship, compared with other plants.In the evolutionary,the same family of plant have more genetic relationship.Meanwhile,closer relationship of the families also get together, suggesting that nitrite reductase mutation in the exolutionary process is small,It is highly conserved.This speculation is supported by using net sever NCBI Blast.4 Functional predictions were carried out in net server Pfam. The results showed that SbNiR was a new member of NiR family, containing ferredoxin domain, [4Fe-4S] domain, It had no singal peptide, but one transmembrane domain in C terminal, indicating that SbNR was acted as a receptor in membrane.