Cloning And Functionnal Analysis Of The MsERF003 From Alfalfa In Carbonate Stress Response

Biological treatment is an effective method to treat soda saline-alkali soil.Alfalfa has strong saline-alkali resistance,therefore,exploring saline-alkali resistance genes thereof and illustrating the potential mechanism within the resistance are beneficial to the breeding of highly alkaline-resistant alfalfa cultivars and their comprehensive utilization in saline-alkali soil bilological treatment.AP2/ERFs transcription factor family is a kind of transcription factor closely related to saline-alkali regulation in plant,which plays important roles in growth and development,metabolism,stress responses,signal transduction and many other functions.Investigation on these transcription factors in alfalfa would be helpful to reveal and illustrate the mechanisms in alfalfa saline-alkali resistance and,this would provide fine saline-alkali resistant genes for crop breeding.In the present study,a gene with an open reading frame of 570 bp in length and encodes 190 amino acid residues from alfalfa,namelyMsERF003,was cloned.The sequence homology betweenMsERF003andMtERF003,a homolog fromMedicago truncatula,was 98.84%.The expression pattern ofMsERF003 under carbonate stress was investigated by qPCR method.The result showed that it was up-regulated in all organs in carbonate treatment,and extemely significant in roots and stems.A vector with GUS gene driven by the promoter of the homologMsERF003,MtERF003,was constructed and,transformation of tobacco was carried out and transgenic plants were obtained.GUS staining analysis showed that the promoter ofMtERF003 was a root-specific in tobacco and only functioned under carbonate stress.A vector with a fusion gene,MsERF003-GFP,was constructed to analyze its subcellular localizationMsERF003 and the results from transient expression showed that MsERF003 was only located in the nucleus.A vector withMsERF003 constructed and transgenic tobacco plants were generated.The saline-alkali resistance of T1 plants was assessed.The results showed that after 8 days treatment with 1.2% carbonate,non-transgenic plants got wilted,withered and yellow,while transgenic plants growed well.During the process of treatment,the total sugar content,proline content,CAT content,APX content,POD content and Na~+/K~+ratio in transgenic plants were significantly higher than those of non-transgenic group.The malondialdehyde content in control group was significantly higher that of transgenic group,and the degradation rate of chlorophyll content was significantly faster than that of transgenic.Expression of stress-resistant response genes in transgenic plants were higher than those in the control group at the later stage of treatment.In conclusion,the study presented herein showed that the expression ofMsERF003 in tobacco improves the saline-alkali resistance of transgenic tobacco andMsERF003 may play an important role in the saline-alkali resistance response of alfalfa.