Cloning And Functional Analysis Of MsHSP70 Gene In Medicago Sativa L.

Heat shock protein(HSP) is a kind of ubiquitous protective protein that plays a crucial role in plant. HSP act as a molecular chaperone participates in cell protection, photosynthesis, genetic breeding, stress tolerance and other processes. We analyzed the characterization and function of Heat shock protein gene MsHSP70 from alfalfa in order to provide candidate gene for genetic improvement and variety breeding in Medicago sativa. The mainly research results are as follows:1. In this study, the homologous cloning method was used in obtaining MsHSP70(GenBank accession No:KJ621409) gene sequence from alfalfa. A full-length 2146 bp fragment containing a 2028 bp open reading frame and encoding a 72.33 KDa polypeptide of 675 amino acids was cloned. MsHSP70 protein is mainly constituted of α-helix and β-sheet. A multiple sequence alignment showed that MsHSP70 shared high sequence identity with Medicago trunctula HSP70(94.47%), Pisum sativum HSP70(89.93%) and Arabidopsis thaliana HSP70(78.72%).2. The results showed that MsHSP70 gene was localized to the mitochondria by subcellular localization experiment and online prediction. A phylogenetic tree analysis revealed that MsHSP70 is classed as plant mitochondrial HSP70. MsHSP70 protein was successfully expressed in E.coli with drive of 35 S promoter and inducement of IPTG.3. The differential expression in alfalfa showed that MsHSP70 was expressed largely in flower, followed by stems and leaves, the least in root. Alfalfa was treated with different stresses, the results indicated that mRNA expression levels of MsHSP70 gene were remarkably induced under heat shock, ABA and hydrogen peroxide stresses. However, its expression did not changed significantly under drought and high salinity stresses.4. The plant overexpression vector of pCAMBIA1302-MsHSP70 was constructed by recombinant DNA technology. Then the MsHSP70 was transformed to Arabidopsis and alfalfa by the Agrobacterium-mediated transformation. The PCR, RT-PCR and Southern blotting analysis from a different perspective were proved that we have obtained transgenic Arabidopsis and alfalfa.5. The mRNA expression of MsHSP70 in transgenic Arabidopsis was triggered remarkably under ABA, drought and salt stresses compared with the wild type. Moreover, both the proline content and activity of superoxide dismutase(SOD) increased significantly in transgenic plants, then on the contrary, the malondialdehyde(MDA) content droped dramatically with ABA and drought treatment, and the relative water content(RWC) was higher than WT under drought condition. There is of no significant difference of physiological index above-mentioned between transgenic and WT plants under salt stress. Meanwhile, the weight and root length of transgenic Arabidopsis seedlings were measured under ABA, drought and salt stresses. Our data displayed that fresh weight and root length of transgenic Arabidopsis seedlings was greater than WT with ABA and drought stresses, then no large difference under salt stress. In summary, the MsHSP70 might play important roles for improving ABA and drought tolerance in the transgenic Arabidopsis.