Cloning And Functional Analysis Of Asparagine Synthetase Gene From Triticale

Objective: Asparagine synthetase, which synthesize asparagine, is an important enzyme inmetabolism of nitrogen cycle in the plant. Expression of Asparagine synthetase gene will increases andAsparagine accumulates more in plant when plants are under drought, salt, mineral element stress andgerms. Asparagine synthetase is a potential stress resistance related enzyme and it expression are inducedby adversity environment factors. To identify Asparagine synthetase gene from hexaploid triticale with awide range of adaptability, and analyze the Asparagine synthetase gene expression characteristic in triticaleunder salt stress.Methods: Based on cDNA-AFLP and silico cloning technology, an asparagine synthetase gene(TriAS) was cloned from triticale, and its biological information was analyzed based on bioinformatics tools.Expression characteristic of the new cloned gene was detected with real-time PCR in triticale varitieswith different salt tolerance ability under NaCl stress. A plant transformation vector TriAS-PZP wasconstructed and transformed into Arabidopsis through infected flowers mediated by Agrobacteriumtumefacien, and was transformed into Nicotiana tabacum by Agrobacterium-mediated leaf disc. Thephysical performance of the wild and transgenic Arabidopsis and tobacco were investigated under salt anddrought stress, and SOD activity and MDA content were detected to infer the probable function of clonedAsparagine synthetase gene.The main results are as follows:⑴Bioinformatics characteristics ofAsparagine synthetase in Triticale.The gene of Asparagine synthetase in Triticale was cloned by silico cloning technology, and it wasregisted as KC193248in GeneBank. The gene encodes a protein of587amino acid with a1761bp openreading frame, and its predicted molecular weight is65.83kD. The putative protein was highly homologousto AS proteins from Triticum aestivum and Gossypium hirsutum. The inferred gene is Asparaginesynthetase gene and named TriAS.The conserved domains of TriAS covered Glutamine transaminasedomain and Asparagine synthetase domain. Phylogenetic analysis indicated that the protein washomologous to AS of Hordeum vulgare.⑵Expression ofAsparagine synthetase gene in triticale under salt stress.The Gene expression in XXHM3with higher tolerance and XXHM5with sensitive to salt stress areall increased first and then decreased under the150mmol/L NaCl stress, and its expression is enhancedobviously more in leaves. The cloned gene expression level in XXHM3is more higher than that inXXHM5in leaves and root. The gene maximum expression appear at36h in XXHM3, and24h in XXHM5under salt stress.⑶Physical performance of transgenicArabidopsis and tobacco under salt and drought stressThe germination rates of transgenic Arabidopsis seeds is86.67%and the increment of the seedlingroot is0.86cm,these numerical value for transgenic Arabidopsis plants were more higher than that of thewild type under salt stress, and MDA content and SOD enzymatic activity are reverse. The performance oftransgenic Arabidopsis are better than the wild type under salt and drought stress. MDA content is less intransgenic plant than in the wide type, and SOD enzyme activity are reverse in the two types plants.Growth performance of transgenic tobacco are better than the wild type under salt and drought stress. BothMDA content and SOD enzyme activity in transgenic tobacco is less than that in the wild type.Conclusion: The gene of Asparagine synthetase in Triticale was cloned, which is encodes a protein of587amino acid with Glutamine transaminase domain and asparagine synthetase domain structure. Theenhancement degree of Asparagine synthetase gene expression is different in the different triticale cultivarswith different salt tolerance ability, and also different in leaves and roots. The transgenic Arabidopsis or tobacco with TriAS grows better than the wild type under the salt or drought stress, The changes of MDAcontent and SOD enzyme activity in the transgenic Arabidopsis or tobacco with TriAS and the control in theexperiment confirm the results. All these results indicated that the Asparagine synthetase gene can enhancethe salt tolerance for triticale.