Salt stress is one of the major abiotic stresses that limit geographical distribution and adversely affect productivity and quality of plants. To cultivate the new varieties of high salt-tolerant plants by transforming salt-related genes into plants will be one of the effective ways to cope with salt stress.Salt stress mainly caused by Na+ ions.The sequesteration of Na+ into vacuole mediated by vacuolar Na+/H+ antiporter is a cost-effective strategy for re-establishing Na+ homoestasis, which can reduce cytosol Na+ content and contribute to osmotic adjustment for water uptake. But the relatively low Vmax of the cloned Na+/H+ antiporters are a limitation for the application of the Na+/H+ antiporter genes for crop molecular breeding.DNA shuffling has been one of the most effective directed evolution methods since reported in 1994. DNA shuffling performed well in the evolution of DNA, protein and even whole organism. In our previous study, Arabidopsis Na+/H+ antiporter was evolved through DNA shuffling combining with the heterologous expression of the shuffled gene products in yeast to screen out novel Na+/H+ antiporter genes, AtNHXS1, which conferring improved activity and enhanced tolerance of yeast to high NaCl concentrations. Yeast complementary test showed that AtNHXS1 conferred one-fold improved NaCl resistance in yeast compared with that of wild type AtNHX1. In this research, the AtNHXS1 was transformed into rice(Oryza sativa L.) and tobacco (Nicotiana benthamiana L.) to further investigate whether this gene can enhance salt-tolerance of transgenic rice plants and tobacco plants.The plant expression vector pCAMBIA1301-35SN-AtNHXS1 was constructed and introduced into the Agrobacterium EHA105. The vector was introduced into rice plants by Agrobacterium-mediated transformation method. After hygromycin screening and PCR test,20 transgenic lines were identified and six transgenic lines were used to do GUS coloring. All these six lines accorded with genetic separation ratio 3:1. Among these, two lines, which were further verified by southern blot, showed that AtNHXS1 was single gene insertions, which were used for further experiments. Real-Time PCR was performed to analyze the transcript level of AtNHXS1 under salt stress. The result showed that the expression levels of AtNHXS1 mRNA were significantly higher after salt treatment in transgenic lines. Under different NaCl treatments, transgenic plants grown significantly better with higher drought weight and fresh weight, compared with that of the wild type plants. Furthermore, overexpression of AtNHXS1 confered rice plants accumulating more Na+, little lower K+ content.Meanwhile, we also transformed AtNHXS1 tobacco by Agrobacterium-mediated leaf disc to further study the salt tolerance ability of this new gene. After hygromycin screening and PCR test, ten transgenic T2 lines were identified, and two lines of which were single gene insertion were used for further salt tolerance assays. Real-Time PCR analyses showed that the expression levels of AtNHXS1 mRNA were significantly higher after salt treatment among different tissues, especially in leaves in transgenic lines. After salt stress, the growth statuses of transgenic lines were dominant better than that of the wild type lines. Under the treatment of 400mM NaCl, the transgenic plants grew well but the wild type plants almost died.Our result suggested that the Na+/H+ antiporter new gene AtNHXS1 could singificantly improve the ability of salt tolerance of rice and tobacco, and could be further applied in the molecular breeding of plant salt tolerance. |