Cloning And Functional Characterization Of MsLEA3-1 Gene From Alfalfa(medicago Sativa L.)

The entire coding cDNA sequence of MsLEA3-1 (Genebank accession No. EU665182) was amplified from alfalfa by reverse transcription polymerase chain reaction (RT-PCR). Open reading frame of MsLEA3-1 was 1314 bp revealed by sequencing and sequence analysis. It encoded a protein of 436 amino acids and without mutation.To test the expression of MsLEA3-1, real-time RT-PCR was performed. Results indicated the transcripts of MsLEA3-1 were much richer in leaves than in both roots and stems of the adult alfalfa. Under NaCl (150mM) treatment, MsLEA3-1 mRNA accumulated quickly, showed two expression maximas and declined to normal level at 24h. In response to ABA (0.1mM) treatment, the mRNA accumulated slowly, reached the maximum by 48 h.Fusion protein expression analysis in Escherichia coli showed that its expression in prokaryotic system could be induced successfully by IPTG.To examine the subcellular localization of MsLEA3-1, the fusion GFP-LEA was constructed and transformed into onion epidermal cells by a gene gun, fusion proteins were examined by a confocal laser scanning microscope. Results revealed that MsLEA3-1 preferentially distributed to nucleus.By inserting MsLEA3-1 into vector pBI121, super expression vector pBI-LEA was constructed successfully and then transferred to tobacco and alfalfa by Agrobacterium mediated transformation system. Twenty-five kanamycin-resistant tobaccos were obtained. Among them, twelve lines were sampled to detect target fragment Results showed that all of them were positive. Southern blot analysis showed that target gene had been integrated into the genomes.Thirteen kanamycin-resistant alfalfa plants were obtained in which four positive plants were detected. RNAi vector pART-F-R of MsLEA3-1 was constructed using pKANNIBAL and pART27 vectors and transformed into tobacco. PCR testing showed that sixteen transgenic plants were obtained.To study the salt tolerance of transgenic tobaccos, NaCl (240mM) stress treatment was performed.The malondialdehyde (MDA) content, electrical conductivity, relative water content (RWC) and proline content were measured. Results showed that the MDA content and the electrical conductivity were both significantly lower in the transgenic tobaccos than wild ones. However,the RWC and proline content accumulated much more in transgenic tobaccos than wild plants. It demonstrates a role for the MsLEA3-1 protein in stress protection and suggests the potential of the MsLEA3-1 gene for genetic engineering of salt tolerance.