A Novel Na+~/H~+Antiporter Gene NHXFS1Obtained By DNA Family Shuffling Coμld Confer Plants More Powerfμl Salt Tolerance

Agricultural productivity, plant growth and ecological environment are severely affected by soil salinity. Many researches are amide toward the breeding of crop cultivars with improved salt tolerance and ecological management which were regarded as an effective way to solve this problem. In order to adapt to the salt stress caused by osmotic stress and ion poison, plant has formed a series of defense mechanisms, mainly including osmotic regulation and reconstruction of the ion balance. To keep the ionic balance, plants were mainly through compartmenting of Na+into vacuolar by the vacuolar Na+/H+antiporter to eliminate the ion poison, this strategy could maintain a lower sodium concentration to protect the enzymes in the cytoplasm and also could reestablish a new osmotic pressure to promote cell water absorption. Many researches showed that overexpression of vacuolar Na+/H+antiporter genes conferred salt tolerance in several transgenic plants, theses results suggested that plant vacuole Na+/H+antiporter genes played a significant role in plant salt tolerance.However, the relatively low and different activity between various types of vacuolar Na+/H+antiporter limited the application of this gene for crop molecular breeding. Creating the new antiporter genes and molecular evolution of the vacuolar Na+/H+antiporter with higher salt tolerance ability are urgent needed for plant molecular breeding of salt tolerance.DNA family shuffling is a method for permutation of natural genetic diversity and a powerful process for directed evolution, which generates diversity by recombination, combing useful mutations for individual gene. It mimics and extends classical breeding methods by recombining more than two parental genes or genes from different species in a single DNA shuffling reaction. It is a powerful tool for rapidly evolving genes, operons and whole viruses for desired properties.In this study, we conducted the rapid evolution of the vacuolar Na+/H+antporter using DNA family shuffling and obtained a novel Na+/H+antiporter gene named NHXFS1with higher ability of salt tolerance.1. Four wide type vacuolar Na+/H+antiporters from Arabidopsis thaliana (AtNHX1),oryza sativa(OsNHX1) and Dendranthema morifolium (DmNHX1) were amplified. Equal amount of these gene fragments were digested with DNase I. The column purified100-200bp fragments were assembled through PCR without primer which were used as the template for further PCR reaction with primers of parental genes and establishing a library of recombind genes. The reassembled fragments of the right size were digested and ligated into yeast expression vector pYPGE15, the constructs were transformed into the double mutant yeast strain Δenal-4::Δnhxl. After plating these transformers on the screening APG plates with increased salt concentrations, a clone which harboring the reassembled gene with higher salt tolerance were obtained and a new Na+/H+antiporter were named NHXFS1.2. Sequencing of NHXFS1and DNA alignment showed that this novel gene has four nucleic acids diversification, G185A,T1059A,T1221G, A1517G, compared with OsNHX1, which resμlt in two amino acid differences G62D, D506G, respectivity.Yeast complementary test showed that the NHXFS1transformed yeast strain could grow in the350mM NaCl APG plates, whiles the AtNHX1, OsNHX1and DmNHX1transformers coμld only grow on100mM NaCl. This resμlt showed that the salt ability of NHXFS1is3.5fold improved in yeast.In the study of subcell localization, confocal microscopy showed that the green fluoresence of NHXFS1-EGFP coincides with the fluorescence of the FM4-64, which indicating that this gene was predominantly located in the membranes of the vacuole and endosomes in yeast.The mutagenesis of NHXFS1and the yeast complementary test of two mutants, NHXFS62and NHXFS506, were conducted. The resμlt showed that the salt tolerance ability of yeast overexpressing of NHXFS62is more powerfμl than that of NHXFS506strain. However, both are weaker than the strain of NHXFS1in the salt tolerance ability. Ion content measurement suggested that Na+and K+in the vacuolar of NHXFS1transformed strain is the highest compared with that of OsNHXl and two mutants.3. The polyclonal antibody against the C-terminal fragment of NHXFS1was prepared. The expression vector pET32a-NHXFS1Antigen containing6×His tag was constructed and were transferred into the E.coli strain BL21(DE3). After IPTG induced, the fusion protein was purified by Ni-NTA superflow Cartridges and was immunized with rabbits for polyclonal antibody preparation. The ELISA results showed that the potency of polyclonal antibody reached1:128000. To identify the antibody specificity, the protein expressed in BL21was analyzed by western blotting. The result indicated that we have obtained high specificity polyclonal antibody which could be used for further study of Na+/H+antiporter.4. In order to test the function of NHXFS1in the plants, the expression vector pGRAB-NHXFSl and pGRAB-OsNHXl were constructed and were transformed into the Agrobacterium AGL1. The transgenic plants were produced by transforming the agribacterium culture into the Arabidopsis with the traditional flower dipping methods. The transgenic Arabidopsis were identified through PCR and the homozygous NHXFS1and OsNHX1lines were obtained for further salt tolerance evalution.After salt treatment, the growth status of Arabidopsis overexprssing NHXFS1is better, with higher height and higher fresh and dry weight, than the plants of overexpressing OsNHX1and wide type Arabidopsis. Real-time PCR and western blotting showed that the NHXFS1transcript and NHXFS1protein overexpressed in the transgenic Arabidosis plants. Furthermore, the NHXFS1transgenic Arabidopsis plants accumulated more Na+,K+and proline.