Cloning Of Glutamine Synthetase Gene And Response To Nitrogen In Sugar Beet(Beta Vulgaris L.)

Sugar beet(Beta vulgaris L.) was an important crop for sugar production all over the world. In northern China, it was an endemic sugar crop. Nitrogen was one of limiting factors for growth and development in sugar beet. Glutamine synthetase(GS) played a central role in nitrogen metabolism in higher plants and was a key enzyme during this process. GS occurred as a number of isoenzyme forms and these GS isoforms were located either in the cytosol(GS1) or plastid(GS2). It was very important to study the structure and function of GS and its expression for realizing highly ammonium assimilation, high yield and good quality in sugar beet. In this study,GS1mRNA , GS2mRNA and GS2 genomic DNA were cloned by RT-PCR from sugar beet, and their structure and function were analyzed by bioinformatics. Finally, the expression of the GS1mRNA and GS2mRNA was tested by semiquantitative RT-PCR under nitrogen and actinomycin D(inhibitor of nucleic acid synthesis) and cycloheximide(inhibitor of protein synthesis) conditions. The main conclusions were showed as follows:GS2 genomic DNA(GenBank accession number: EU558132) was firstly isolated from Beta Vulgaris L by PCR and its sequence was 6144bp in length and contained 13 exons and 12 introns. Among these introns, the longest one was 1576bp and the shortest one was 89bp in length.The known genes of GS1mRNA(GenBank accession number: AF343667) and GS2mRNA (GenBank accession number: AY026353) were cloned from sugar beet by RT PCR. GS1cDNA sequences obtained were 1071bp in length, and the sequence similarity between this cDNA and the known GS1mRNA was 99.81%, and there were 2 bases difference. The cDNA encoded 356 amino acids and the amino acid sequences showed high identity with other plants GS1 genes. GS2cDNA sequences obtained were 1296bp in length, and the sequence similarity between this cDNA and the known GS2mRNA was 99.92%, and there was 1 base difference. The cDNA encoded 431 amino acids and the amino acid sequences showed high identity with other plant GS2 genes.GS1 and GS2 protein structure and function were predicted with different bioinformatic methods. The results of primary structure analysis showed that GS1 and GS2 were soluble and hydrophobic, and PI and Mw of GS1 were 5.30 and 39092Da while that of GS2 were 5.91 and 47422Da. The GS amino acid sequences derived from sugar beet have extensive homology with GS from other higher plants. The phylogenetic tree displayed by DNAMAN 6.0 soft showed that the GS1 from Beta Vulgaris had closer relationship with Elaeagnus umbellata than with other higher plant and belonged to cytosolic GS family. And the GS2 had closer relationship with Spinacia oleracea than with other higher plant and belonged to chloroplastic GS family. Secondary structure analysis indicated that GS belonged to mixed protein class. The alpha helix and extended strand of GS1 were about 24.72% and 16.01%, and that of GS2 were about 24.13% and 16.01%. 3D structure constructed by homology method in Geno3d showed that there were 10 helices and 19 strands in GS1, and 12 ones and 10 ones in GS2. Sequence and structural analysis showed that GS protein had Gln-synt conserved domains contained beta-Grasp domain from N-terminal and catalytic domain from C-terminal.The effects of different nitrogen ratios(NO3--N:NH4+-N) on the expression of GS1 and GS2 gene in sugar beet were similar to the changes of GS activity. The expression of GS gene was activated best when the ratio of NO3--N and NH4+-N was 80:20 and 50:50. But that was worst when the ratio of NO3--N and NH4+-N was 0:100. Namely, the expression of GS gene was activated with mixed nitrogen supply, but decreased when the sole nitrogen source was used. And the single nitrogen source of NO3--N accelerated the expression of GS gene better than that of NH4+-N.The effects of actinomycin D and cycloheximide on the expression of GS gene were similar to GS activity changes after induction with nitrogen. The GS activity was inhibited by actinomycin D and cycloheximide. The expression of GS mRNA was lowered by actinomycin D, but promoted by cycloheximide at a certain extent. Therefore, this study indicated that the gene expression of GS in sugar beet induced by nitrogen was regulated and controlled not only at transcriptional and translatable levels, but also on the level of GS activity.