The Influence Of Alfalfa Ethylene Response Factors On Salt-tolerance Of Plants And Characteration Of Mswrky56Gene In Alfalfa (Medicago Sativa L.)

Ethylene response factors (ERFs) are plant-specific transcription factors which involved in plant growth, development and response to stresses. AP2/ERF domain of Arabidopsis has been used as probe to search the medicago protein database in national center of biotechnology information, and then gene-specific primers have been designed base on the amino acid and nucleotide sequences to isolate ERF genes from alfalfa. Two ERF orthologues have been isolated from alfalfa, named as MsERF8and MsERFll respectively. Additionally, A cDNA sequence of alfalfa WRKY transcription factor gene was isolated by electronic cloning and RT-PCR, named MsWRKY56, and bioinformatics analysis has been performed. All the results are as follow:1. The MsERF8cDNA has an open reading frame (ORF) of603bp, encodes a nuclear protein of201amino acids and the ORF of MsERF11gene was804bp for268amino acids. The theoretical isoelectric point (pI) and molecular weight (Mw) of MsERF8and MsERF11protein was6.91(pI),22.88kDa (Mw), and7.74(pI),29.05kDa (Mw). They have alpha helix, extended strand and random coil in their secondary structure. Most regions of MsERF8and MsERF11were hydrophilic. Phylogenetic tree analysis of MsERF8and MsERF11and other AP2/ERF proteins revealed that MsERF8and MsERF11belong to the ERF sub-family. 2. The genomic sequences of MsERF8and MsERF11have been obtained using alfalfa genomic DNA as temple, sequence analysis revealed that all these two genes have no introns in their structure. The promoter of these two genes have been isolated by using TAIL-PCR, the cis-element analysis revealed that MsERF8promoter contains ERE、MES、 TATC-box in its promoter region, and MsERF11promoter contains ABRE、methyl jasmonate response element、and lots of elements associate with light response.3. The expression patterns of MsERF8and MsERF11in different tissues of mature alfalfa plants and the effect of abiotic stresses and hormones on expression of these two genes were detected by semi-quantitative RT-PCR and real-time quantitative PCR. MsERF8was strongly enriched in roots and leaves compared with stems, flower buds and flowers, and a higher level of MsERF11expression was observed in leaves than was observed in roots, stems, flower buds and flowers in mature alfalfa plants. All these two genes were induced by NaCl、PEG6000、Al2(SO4)3and diverse hormones, IAA could induced the expression of MsERF11but has no effect on the expression of MsERF8. Transient expression vectors of these two genes have been constructed to demonstrate the subcellular localization of their productions, the onion epidermis was used as a recipient host and the transformation were performed using a gene gun, the results indicated that all the productions of these two genes were located in nucleus.4. The ORF of MsERF8has been transformed to tobacco and the ORF of MsERF11has been transformed to Arabidopsis using Agrobacterium-mediated method. The germination rate and relative water contents of transgenic tobacco and Arabidopsis plants were significantly higher than wild-type plants after NaCl treatment (P<0.05), the MDA content of transgenic plants were significantly lower than wild-type tobacco plants (P<0.05), the free proline content of transgenic tobacco plants were significantly higher than wild-type plants (P<0.05). Additionally, growth inhibition was greater in non-transgenic Arabidopsis seedlings than in transgenic Arabidopsis seedlings after NaCl treatment. All of these results demonstrated that the transgenic plants have a high level of salt-tolerance compared with the wild-type plants.5. The cDNA sequence of MsWRKY56was1267bp long and contains an ORF of951bp for317amino acids. The pI/Mw of MsWRKY56protein was6.91and22.88kDa. It has alpha helix, beta fold in its secondary structure. Most regions of MsWRKY56were hydrophilic. The MsWRKY56protein has no signal peptide and locate in nucleus.All of our results provide the foundation for further study of the structure and function of ethylene response factors and WRKY transcription factors in alfalfa, and supply candidate genes for enhancing tolerance to salinity in breeding crops.