Cloning And Expression Analysis Of Glutamine Synthetase Gene GS From Eichhornia Crassipes

Glutamine synthetase (GS) plays a central role in nitrogen metabolism in all plants plants, which is a key enzyme during ammonia assimilation. Plant GS occurs as a number of isoenzyme forms and these GS isoforms are located either in the cytosol (GS1) or plastid (GS2).Eichhornia crassipes can grow rampantly in eutrophic water growth, which made the ecological system out of balance. But E. crassipes can excessively absorb the nitrogen and phosphorus, so E. crassipes can be used to purify water.Based on amino acids sequences of glutamine synthetase from other plants, such as soybean, Triticum aestivum and so on, we designed two pairs of degenerate primers to clone the conserved region of glutamine synthetase gene from E. crassipes, and then used RACE technology to amply three full-length cDNAs. The isolated EcGSla, EcGS1b and EcGS1c encoded356,354and356amino acids, respectively. Sequence and structural analyses showed that all EcGS1a, EcGS1b and EcGS1c proteins contained putative ATP-binding region, N-glycosylations, N-glycosylations, GS beta-Grasp domain and GS catalyticdomain that are conservative with other plant GS. The prediction of sub-cellular localization showed that EcGS1a, EcGS1b and EcGS are all cytosolic glutamine synthetase. The amino acid sequences deduced from EcGS1a, EcGS1b and EcGS1c gene sequences have extensive homology with GS1from other higher plants. The phylogenetic tree displayed that the EcGS1a, EcGS1b and EcGS1c was the most closed to GS1from Hevea brasiliensis.The transcription of three EcGS1genes and GS activity affected by three different forms of nitrogen were analyzed. In root, the transcriptions of EcGS1a and EcGS1c were mainly affected by nitrate, the expression of EcGSlb transcription was almost not affected by forms of nitrogen, GS activity was inhibited by nitrogen starvation and ammonium, but is not inhibited with the time of treatment. In leaf, the transcriptions of EcGSla, EcGSlb and EcGS1c were enhanced under three forms of nitrogen, the transcription of EcGS1a was the most significant affected by nitrogen starvation, the transcription of EcGS1b and EcGSlc were the most significant affected by nitrate, GS activity was not affected by nitrogen starvation, GS activity was significantly enhanced after2h ammonium nitrogen and4h nitrate nitrogen treatment.