Research On The Expression And Salt Tolerance Of SsNHX1 Gene In Medicago Sativa L.

In this study, a putative complete vacuolar-type Na+/H+ antiporter gene (SsNHX1) gene was isolated from the halophyte Salsola soda by using the rapid amplification of cDNA ends (RACE) method. The SsNHX1 was constructed into a plant expression vector and transformed to alfalfa(Medicago sativa L.). And we studied on the salt tolerance for transgenic plants.The sequence of SsNHX1 was 1934 bp in whole length which had one open reading frame (ORF) of 1680 bp, encoding 559 amino acid residues. Highly conserved regions of plant vacuolar Na+/H+ antiporter, including amiloride-binding domain, NHE (Na+/H+ exchanger) domain and 12 transmembrane segments, were found in the deduced amino acid sequence of SsNHX1. Multiple alignment of vacuolar Na+/H+ antiporters showed SsNHX1 shared high identity with vacuolar Na+/H+ antiporters of Kalidium foliatum, Atriplex gmelini, Medicago sativa, Gossypium hirsutum, Arabidopsis thaliana, Oryza sativa, Hordeum vulgare, and Zea mays, the similarity was 90%,87%,77%,76%,75%,74%,73%, and 72%, repectively. Phylogenetic relationship analysis indicated that SsNHX1 had closer affinity with vacuolar Na+/H+ antiporter group, while they were far with plasma membrane Na+/H+ antiporter group. Meanwhile, the SsNHX1 was more closely related to halophyte, Kalidium foliatum and Atriplex gmelini, and it's relationship was far related to glycophyte. The expression of SsNHX1 could improve growth rate of transgenic yeast under salt tolerance. Additionlly, the expression of SsNHX1 could increase the germination rate of T1 seeds of transgenic tabacco, and increase the height and productivity significantly compared with control plants. These results confirmed the salt-tolerant function of SsNHX1. Taken together, these results suggest that SsNHX1 is a new member of the vacuolar Na+/H+ antiporter family, and the overexpression of this gene could obviously enhanced the salt tolerance ability in plant. The genotype of alfalfa was the key factors in genetic transformation, therefore, there were only a few cultivars suitable for the establishment of high efficient regeneration system. In this study,13 main cultivars were selected for expriment among domestic and foreign species. The experiment showed that Gong-Nong 1 was the best genotype for transformation by comparing the inducing capacity. And based on this conclusion, a high efficiency Agrobacterium-mediated genetic transformation of alfalfa system was established and optimized with leaf of Gong-Nong 1 as explants. Moreover, we have successfully transferred the vacuolar Na+/H+ antiporter gene of Salsola soda into alfalfa genome. The Southern blot analysis showed that there were 6 independent transformed lines were positive. Finally, we obtained 203 positive plants in this study.The six independent transformation lines were taken as material for salt tolerance test. The Northern-blot demonstrated that the expression of SsNHX1 driven by the stress inducible promoter was increased with the treating salt concentration. Salt-stress test showed that the salt-tolerant ability of transgenic alfalfa was enhanced up to 400 mM NaCl compared to control plants died at the stress condition of 200 mM NaCl. And there were higher Na+, K+ content in transgenic plants. The physiological analysis showed that the transgenic alfalfa caused less damage under salt stress compare to control plants. For example, the changes trend of the superoxide dismutase (SOD) activity of transgenic plants was smaller than that of control plants, and proline content was smaller too. In addition, the changes trend of the rate of electrolyte leakage (REL) of transgic plants was also smaller than that of control plants. All these results indicated that the expression of SsNHX1 was much helpful for transgenic plants to resistant to the salt stress, and it could enhance the salt-tolerant ability of transgenic alfalfa effectively.