Expression And Salt Tolerance Analysis Of A Novel Evolved Na~+/H~+Antiporter Gene NHXFS1in Tobacco (Nicotiana Benthamiana L.)

Salt stress is one of the major abiotic stresses that limites the plant growth and causes crop failure. The injury caused by salt stress mainly come from high concentration of Na+ions in soil. The high concentration of Na+ions in cell can result in physiological drought and ion toxicity, which could break metabolism balance of plants and then against the plant growth and development.Plant tonoplast Na+/H4+antiporter is a kind of transmembrane protein, which plays an important role in salinity tolerance. The sequestration of Na+ion into vacuole mediated by tonoplast Na+/H+antiporter is an effective strategy to protect plants from salt stress, which can reduce cytosol Na+content and contribute to osmotic adjustment for water uptake.NHXFS1is a novel tonoplast Na+/H+antiporter gene which was previously obtaine from AtNHX1, OsNHX1and DmNHXl by DNA shuffling in vitro in our lab. The open reading frame of NHXFS1is1,608bps which encodes a protein of535amino acids. Sequence comparison showed that there are four nucleic acids mutations between NHXFS1and OsNHX1, which result in two amino acid substitutions. Yeast complementary test indicated that NHXFS1could enhance the salt tolerance ability of yeast to high concentration of NaCl.To further study the expression pattern and salt tolerance ability of NHXFS1in plant, we constructed plant expression vector pCAMBIA1301-35SN-NHXFS1and transformed into tobacco by Agrobacterium-mediated leaf disc method. Fifteen transgenic lines were obtained after hygromycin B screening and PCR identification. Southern blotting analysis with NHXFS1probe indicated the NHXFS1gene was integrated into tobacco genome successfully. According to segregate ratio, transgenic tobacco line with single copy was inbreeded to homozygote and used for further study. RT-PCR analysis showed that the NHXFSl gene was expressed in tobacco normally. The result of Real-time PCR showed that the transcript level of NHXFS1was significantly higher in different tissues after200mmol/L NaCl treatment, which increased1.01,0.6,1.79fold in root, stem and leaf, respectively. The result of salinity tolerance test demonstrated that the transgenic seeds could still germinate in MS plate with400mmol/L NaCl, but the controls could not. Also, the growth status of transgenic plants were better than that of controls in soil contained high NaCl concentration. When NaCl concentration reached to300mmol/L, the wild-type plants were nearly wilting, while the transgenic ones were still vital. Compared with that of the wild type plants, the transgenic plants accumulated more Na+, chlorophyll and proline after salt stress.All the results suggest that this novel gene NHXFS1could be applied in plant salt tolerance engineering to improve plants salt tolerant ability. And it also manifested that it is available strategy to develop novel genes by DNA family shuffling method for improving plant salt tolerance.