Study On Glutamine Synthetase Genes Cloning And Over-expression Of Ramie (boehmeria Nivea L.)

The mineral nutrients nitrogen is essential for higher plants growth, development and a majorlimiting factor in crops yield and quality. Glutamine synthetase (GS) plays fundamental roles in higherplants nitrogen primary assimilation and is regarded as the “hinge” over which inorganic nitrogen isconverted into organic form and transformed into plants. GS isoenzymes have essential effects to thenitrogen absorption, assimilation and use efficiency. Therefore, GS isoenzymes have critical functionsin growth and development, yield and quality of crops. According to their localization with the cell, GSisoforms include cytosolic (GS1) and plastid (GS2). The cytosolic isforms correlate with assimilation ofNH+4reduced from NO-3and re-assimilation of NH+4released from various metabolic pathways andGS2mainly exist in leaves with abundant chloroplast for incorporation of ammonium fromphotosynthesis and photorespiration. Ramie (Boehmeria nivea L.) is world-famous for its excellent fiberand commonly known as “China grass”. Simultaneously, ramie is regarded as ingredients for forageprotein due to equal nutritional value with alfalfa (Medicago sative), high protein and amino acidcontent, reasonable nutrition component and high biomass. In addition, ramie has special characteristicof growth having high rapid growth rate with4-6cm per day at vegetative stage. However, there haslittle or no physiology and molecular document been invested in understanding of ramie nitrogenassimilation and metabolism, and no functional genes in ramie nitrogen pathway have been excavatedand utilized. With the aim of gaining new insights in this area, especially at molecular levels, the focusof our study was first isolation and characterization of ramie GS gene families, and comparativelyanalyzed of GS gene families sequences, GS gene families expression pattern at different tissues anddevelopment stages and higher plants GS phylogeny. In addition, the over-expression plant vector oframie BnGS1-2gene was constructed according to homologous recombination technology andtransgenic tobacco was obtained by “leaf-disk” transformation method. Therefore, the investigation ofthe effects of BnGS1-2over-expression in improving the nitrogen absorption, assimilation and useefficiency provided theoretical basis at molecular level for ramie GS function and nitrogen assimilationpathway, and material basis for utilization of ramie GS genes. The main study results were indicated asfollows:（1）Four genes of ramie GS gene families were first isolated from cultivator “Zhongzhu No.1”,two genes encoding cytosolic GS1and the other two encoding plastid GS2, named BnGS1-1, BnGS1-2,BnGS2-1, BnGS2-2, respectively. In addition, the two isolated BnGS2were allele genes, identified byTaq Ⅰ digestion of target genes in self-bred progenies, from ramie cultivator “Zhongzhu No.1”.（2）Sequence and structural analysis showed that the cDNAof BnGS1-1gene with length of1205bp including a1071bp ORF region encoded polypeptide of356amino acids; the cDNA of BnGS1-2gene also encoded356amino acid polypeptide with length of1222bp including a1071bp ORF region.Two BnGS2allele genes encoded polypeptides of430amino acids with1340bp length including a 1293bp ORF region and a transit peptide. The diversity of nucleotide in11sites between BnGS2allelegenes resulting into amino acid residues substitution at site195and382. Four genes of ramie BnGSgene families contained beta-Grasp and catalytic functional domains which were belonged to Gln-syntdomain and conservative with other plants GS. The residues in site56,92,249,297of ramie BnGSgenes and in306,371of BnGS2allele genes were Asp, Cys, His, Glu, Cys and Cys respectively.（3）Comparison of nucleotide and amino acid sequences showed that all ramie BnGS genefamilies shared very similar percentage in identity ranging from77.25-91.57%at protein sequence andat nucleotide levels with71.15-79.37%. The phylogenic analysis showed discrepant evolutionrelationship of ramie cytosolic GS genes with BnGS1-1grouped into monocots and BnGS1-2was asister to dicots. Simultaneously, the ramie BnGS1-2and BnGS2genes were similar with alfalfa(Medicago sativa), soybean (Glycine max) and bean (Phaseolus vulgaris) in phylogenic relationship.（4）The expression patterns of ramie BnGS gene families at different tissues and developmentstages were investigated by fluorescence quantitative real-time PCR. The cytosolic and plastid BnGSgenes exhibited non-organ-specificity expression patterns but displayed very different transcriptionalconcentration. The main transcriptional tissue of BnGS2was leaves and gene expression levels variedalong with different development stages. BnGS1-1relative expression levels in leaves, roots and phloemwere higher than those in stems and xylem at all development stages. However, transcriptional levelswere remarkable reduced in leaves at mature stage. BnGS1-2relative expression levels weresignificantly triggered specifically in the phloem and xylem suggesting it has a primary role in ramiefiber development. On the other hand, BnGS1-1and BnGS1-2displayed high relative expression levelsin roots at all development stages. Therefore, the specificity in expression intensity rather than organ oframie different BnGS genes, may be a main factor to regulate nitrogen assimilation and metabolism inthe process of growth and development.（5）The ramie BnGS1-2gene was ligated properly with plant expression vector pBI121toconstruct plant over-expression vector according to homologous recombination technology. Theover-expression vector was transferred into tobacco through Agrobacterium tumefaciens LBA4404using “leaf-disk” transformation method. Transgenic plants were obtained by Kana screening and DNAPCR determination. Q-PCR analysis showed BnGS1-2mRNA was detected in all transgenic tobacco T1plants. The GS enzyme activity in transgenic plant was detected at1-2times higher than wild type. Thetransgenic plants with over-expression BnGS1-2gene were significantly enhanced plant height, freshweight and leaf area suggesting BnGS1-2over-expression is able to promote the growth of transgenicplants. In addition, compared with wild type, the soluble protein of transgenic plants was remarkableenhanced up to92.02%, the total nitrogen content increased but did not reach significant level. TheBnGS1-2transgenic plants also exhibited an remarkable reduce of NH+4content, while the NO-3contentstill maintained the same levels with wild type. These results suggested that BnGS1-2over-expressionenable the transgenic plants to facilitate the nitrogen absorption, assimilation and use efficiency tosustain plants fast-growing demand for nitrogen nutrition. Therefore, the ramie BnGS1-2would be anexcellent gene resource for improvement of plants nitrogen utilization efficiency and crops agronomic traits.