Functional Research Of Asparagine Synthetase Gene AS In P. Simonii×P. Nigra

Asparagine synthetase (asparagine synthetase-B, AS-B, EC 6.3.5.4) was a critical enzyme of nitrogen assimilation in plants involved in transport and storage of organic nitrogen. So the functional study of genes encoding the enzyme played an important role to improve nitrogen use efficiency of crops or trees. In this study, we cloned the AS genes in P.simonii ×Pnigra and analysed expression patterns of daily rhythm, continuous light/dark 48h treatment, different nitrogen concentrations and MSX treatment. We constructed plant expression vector and infected 84K poplar. The plant morphology and physiology indicators in nitrogen metabolism were determined to learn more about the function of the gene. The main conclusions were as follows:(1) We obtained PnAS1, PnAS2 and PnAS3 by homologous cloning method in P. simonii×P. nigra, length of 1770bp,1755bp and 1764bp, and successfully submitted NCBI database that accession number were KT161263, KT161264 and KT161.(2) The growth of E. coli auxotroph ER transformed with the empty vector was very weak in a medium without Asn that almost no change in the growth curve. While the E. coli auxotroph ER strain transformed with pET-14b-PnAS1, pET-14b-PnAS3 and pET-14b-PnAS2 could grow in M9 medium without Asn. Therefore, the three genes had function and could recover E. coli auxotroph ER strain.(3) Organization-wide expression analysed that PnAS1and PnAS2 were mainly expressed in shoots. And three genes were transcribed in root.(4) PnAS3 gene transcript level was lower in shoots. In L1, the fluctuation PnAS2 was remarkable which was the mainly gene, whilePnAS1 gene in LI was stable. In L2, the trend of PnASland PnAS2 as similar that the expression level in dark was slightly higher than in light. In L3, level of PnAS2 in light was slightly higher than in dark in L3.(5) PnAS1 and PnAS3 were suppressed by the continuous light 48h and the dark-induced phenomena, the level of transcription of two genes under dark conditions was 50-200 times than in light conditions, PnAS2 gene suppressed by the light and not effected by dark.(6) PnAS1 was inducedby NO3- firstly (3h), followed by NH4+(72h) in L1. In L2 leaves, two genes were inhibited. PnASl and PnAS3in root were strongly induced that transcription level of PnAS3 was 18,000 times higher than WT plants. Therefore, when supplying of nitrogen, the root was the main part of the induced AS gene. MSX experiment confirms that expression of PnAS3 gene was induced by Gin or Gin derivatives, but not directly to the gene.(7) Under 0.05mM NH4NO3 condition, NUE of transformants 3,5 and 7 increased significantly; biomass and leaf area were all higher than WT plants. Beside, GOGAT activity of transformants 4,5 and 7, was also higher than wild-type plants.In summary, the three AS genes were able to recover to E. coli auxotroph ER strain which was influenced by the light/dark, nitrogen. Under low nitrogen stress conditions, transgenic 84K poplars exhibited in higher nitrogen utilization efficiency, biomass, amino acid and other more advantages, so the transgenic 84K poplars had a significant resistance to nitrogen stress.