Cloning of an RNA-binding Protein Gene from Alfalfa and Expression in Tobacco

Based on an expressed sequence tag of Medicago sativa L.,a full length of 1551 bp cDNA was isolated using RT-PCR and RACE-PCR techniques.The gene was named MsRBP(GenBank accession No.JN986878.1).Sequence analysis revealed that MsRBP contained a maximum open reading frame of 1230 bp and encoded a 409 amino acids protein.The putative amino acid sequence had high identity with RNA binding proteins from Medicago truncatula,Glycine max,and Arabidopsis thaliana.Real-time quantitative PCR showed that the transcription level of MsRBP was up-regulated in NaCl,ABA,and PEG stress treatments.It indicated that MsRBP probably played an important role in the tolerance of abiotic stress.MsRBP gene was transferred into tobacco with leaf disc method by Agrobacterium LBA4404 and expressed under the control of CaMV 35S promoter.The transgenic plants were selected on MS medium supplemented with kanamycin and analyzed by PCR,RT-PCR and GUS assay.These results showed that MsRBP was integrated into tobacco genome and expressed in transgenic tobacco.