Gene Cloning And Bioinformatics Analysis Of Nitrate 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. Nitrate reductase was a key and limited enzyme in nitrogen metabolism. The functional study of nitrate reductase played a central role in increasing sugar beet yields. At present, the main studies of sugar beet nitrate reductase were focused on dynamic changes of nitrate reductase activities and regulations of nitrogen on nitrate reductase activities. The researches of nitrate reductase structure and characteristics were reported few. In this study, SbNR gene was cloned by RT-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 nitrate reductase could be revealed from the enzyme molecular structure. The results were as follows:1 SbNR gene was firstly cloned from sugar beet by RT-PCR and 5'/3'-RACE. The accession number was EU163265. The full length of SbNR was 3247bp and encoded 905 amino acids. The cDNA and amino acid sequences showed high identity with other plant NR genes. Sugar beet NR genomic DNA was isolated by PCR and its sequence contained 4 exons and 3 introns. The accession number was EU571714. The full length of genomic DNA was 6346bp, and the lengths of introns were respectively 197bp, 1088bp and 2343bp.2 Protein structure was predicted with different methods. The results of primary structure analysis showed that SbNR was soluble and hydrophobic, with a pI (isoelectric point) of 6.09 and MW (molecular weight) of 102kDa. Secondary structure analysis indicated that SbNR belonged to mixed protein class. 3D structure was constructed by homology method.3 Functional predictions were carried out in net server. The results showed that SbNR was a new member of NR family, containing molybdopterin-binding domain, cytochrome-binding domain, FAD-binding domain and NAD-binding domain. It had no singal peptide, but one transmembrane domain in C terminal, indicating that SbNR was acted as a receptor in membrane.4 The specific DNA sequences of nitrate reductase were selected as probe to detect gene copy numbers of NR in sugar beet genomic DNA. The genomic DNA was digested by four restriction enzyme(EcoRâ… ,Hindâ…¢,BamHâ… ,Draâ… ). Digested bands separated by gel electrophoresis were transferred in situ to nylon membrane. Hybridized signals were obtained by immunological detection. Four hybridized signals appeared at EcoRâ… digested bands, and MW of signal bands were between 3~5kb. One hybridized signal both appeared at Hindâ…¢and Draâ… digested bands. Two hybridized signals appeared at BamHâ… digested bands, and MW of one signal bands was more than 6kb. These results indicated that NRcDNA amplicated by RT-PCR was the expression products of sugar beet genomic DNA, and gene copy number of NR in sugar beet genomic DNA was single- or low-copy.