| Soil salinity is a major abiotic stress factor in limiting plant growth and crop productivity. To cope with salt stress, plants have developed several adaptive mechanisms including osmotic adjustment and ion homeostasis. The sequesteration of Na+ into vacuole mediated by vacuolar Na+/H+ antiporter is a cost-effective strategy for re-establishng Na+ homoestasis, which can not only reduce cytosol Na+ content, but also contribute to osmotic adjustment for water uptake. The overexpression of vacuolar Na+/H+ antiporter gene conferred salt tolerance in several transgenic plants suggested that plant vacuole Na+/H+ antiporter genes play a significant role in plant salt tolerance.DNA shuffling is a fast and useful molecular biological approach for the directed evolution of protein, enzymes, antibody etc, which has a broad prospects for producing new enzymes or proteins with enhanced activity, altered substrate specificity or improved function.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. In this study, we applied DNA shuffling methodology combined with the heterologous expression of the shuffled gene products in yeast to screen novel Na+/H+ antiporter genes conferring improved activity and enhanced tolerance of yeast to high NaCl concentrations. Meanwhile, we further study the structure-function relationships of the vacuolar Na+/H+ antiporter.Our research can be summarized as follow:1. DNA shuffling of Na+/H+ antiporter gene AtNHXlThe wild-type AtNHXl gene was fragmented with DNasel. The column purified 100-200bp fragments were assembled through PCR without primer to a single gene product of the correct size. After being ligated into the yeast expression vector pYPGE15 and then introduced into the yeast double mutant strain W303-1BΔena1-4Δnhx1, the shuffled gene library was expressed and screened on the APG medium supplied with 100mM NaCl at pH5.5. With the NaCl selective pressure, one clone grown best was selected after the second selection. The shuffled gene expressed in this yeast clone was named AtNHXSl2. Sequence analysis of shuffled Na+/H+ antiporter gene and biochemical characterization.The comparison of AtNHXSl with AtNHXl showed that the AtNHXSl gene has seven nucleic acids mutations and forty-two nucleic acids deletions, which resulted in the presence of a premature termination code. In the amino acid sequence of AtNHXSl, there are four substitutions L29P, S158P, Y241P, F242L, and there is one truncation of 296 amino acids in the C terminus.Yeast complementary test showed that AtNHXSl conferred one-fold improved NaCl resistance in yeast. Furthermore, the yeast strain harboring the shuffled Na+/H+ antiporter gene accumulated more Na+ and slightly more K+ than that expressing AtNHX1.Confocal microscopy showed that, similar to that of AtNHX1-GFP, the fluorescence of the GFP-tagged AtNHXS1 was predominant in membranes of the vacuole and endosomes in yeast.The intact vacuoles were isolated from the yeast expression AtNHXl or AtNHXSl, and the cation transport activities were determined though monitoring the fluorescence quenching of acridine orange. The shuffled Na+/H+ antiporter displayed appropriately 1-fold higher Vmax of the Na+/H+ exchange and weak increased K+/H+ exchange across the yeast vacuole membrane, thus indicating that the novel Na+/H+ antiporter gene possesses enhanced sodium transport activity.3. Mutagenesis and deletion of AtNHX1 and yeast complementary test of those mutants.AtNHX1 with the mutation L29P, or the mutation S158P, or with a 296 amino acids C-terminus deletion were constructed through PCR. After being confirmed by sequencing, the three AtNHXl mutants were introduced into the yeast mutant strain separately. The yeast complemetary test showed that the expression of the AtNHX1 mutations L29P, S158P and the C-terminus truncation displayed similar enhancement of tolerance to NaCl, KCl, LiCl as well as hygromycin B compared with that of the novel AtNHXS1.4. The construction of the plant expression vector and transgenic Arabidopsis The plant expression vector pCAMBIA1301-35SN-AtNHX1 and pCAMBIA1301-35SN-AtNHXS1 were constructed and introduced into the Agrobacterium GV3101. The transgenic Arabidopsis were produced through floral dipping methods. And several homozygous lines transformed with AtNHX1 or AtNHXS1 were selected for the further salt tolerance evaluation.5. The molecular characterization and investigation of the salt tolerance of the transgenic ArabidopsisThe transgenic Arabidopsis were identified through PCR and PCR-southern bloting, which showed that the AtNHX1 or AtNHXS1 were integrated into the genome of the Arabidopsis. Realtime RT-PCR indicated that the NHX1 expresssed high in the transgenic Arabidopsis. Under treatment with 200mM or 300mM NaCl, transgenic Arabidopsis grown significantly better compared with the wild type plants, and the Arabidopsis expressing AtNHXS1 grew better than that expressing AtNHX1, with higher drought weight and fresh weight. Furthermore, overexpression of AtNHXS1 confered Arabidopsis plants accumulating more Na+, K+ and proline in the shoot compared with that of AtNHX1 gene.In conclusion, we used DNA shuffling methodology combined with a yeast high throughput expression system to evolve the plant Na+/H+ antiporter gene AtNHXl and obtained one novel Na+/H+ antiporter gene AtNHXS1.The novel gene expressed protein was localized in yeast vacuole, and displayed a higher Na+/H+ transport activity as well as a moderate improvement in K+/H+ transport activity, that enabled yeast to accumulate more Na+ and K+ in vacuole and conferred increased salt tolerance. Overexpression of AtNHXS1 also confered transgenic Arabidopsis stronger salt tolerance compared with that of AtNHX1. This study supply the new information on the further understanding of struction-functional relation of the plant Na+/H+ antiporter gene, and it is possible for the novel Na+/H+ antiporter gene to be applied in the molecular breeding of plant salt tolerance.
|
PDF Full Text Request