Abstract：Plant tonoplast Na+/H+ antiporter comparting sodium into vacuolar plays an important role in salinity tolerance. NHXFS1 is a novel tonoplast Na+/H+ antiporter gene which is obtained from AtNHX1 of Arabidopsis thaliana, OsNHX1 of Oryza sativa and DmNHX1 of Flos chrysanthemi by DNA family shuffling in vitro. To study the expression pattern and salt tolerance ability of NHXFS1 in plant, we transformed NHXFS1 into tobacco (Nicotiana benthamiana) by Agrobacterium-mediated leaf disc method, the transgenic plants were screened with hygromycin B and identified by PCR and Southern blot. 15 NHXFS1 positive plants were identified from 43 hygromycin-resistant tobacco plants that we had gotten. Southern blotting analysis with NHXFS1 probe indicated that the NHXFS1 gene was integrated into tobacco genome. According to segregate ratio, transgenic tobacco line with single copy was inbreeded to homozygote. The result of salinity tolerance test demonstrated that the growth vigor of transgenic plants under high salt stress was better than that of the control ones. The transgenic seeds could still germinate in MS plate with 400 mmol/L NaCl, but the controls could not. Also, the growth statuses of transgenic plants were better than that of controls in soil contained high NaCl concentration. When NaCl concentration reached 300 mmol/L, the wild-type plants were nearly wilting, while the transgenic ones were still vital. Results showed that the transgenic plants could accumulate more proline and Na+ ion. RT-PCR analysis showed that the NHXFS1 gene was expressed in tobacco. The result of Real-time PCR showed that the transcript level of NHXFS1 was significantly higher in different tissues after 200 mmol/L NaCl treatment, which increased 1.01, 0.60 and 1.79 fold in root, stem and leaf, respectively. The data suggest that this novel gene NHXFS1 can be applied in plant salt tolerance engineering and molecular breeding. And it also manifest that it's available to develop novel genes improving plant salt tolerance by DNA family shuffling method
