Employing DNA Family Shuffling For Creating A Novel Strong Plant Na~+/H~+ Antiporter Gene

Soil salinity is a major abiotic stress factor that impacts plant growth and productivity. To avoid the toxic effects of salt, plants especially halophytes have developed a mechanism to sequester Na+ into vacuoles through Na~+/H~+ antiporters. The compartmentation of Na+ in vacuoles averts the deleterious effects of Na+ in the cytosol, while mediating vacuolar osmotic adjustment that facilitates water uptake into cells, thereby facilitating the improvement of plants adaptation and tolerance to salinity. The vacuolar Na~+/H~+ antiporter is an important determinant in controlling plant salt tolerance as evidenced by the enhanced tolerance to salt stress in various plant species overexpressing Na~+/H~+ antiporters. However, the identified Na~+/H~+ antiporters have the problems of the relatively low Na~+/H~+ exchange efficiency and low salt tolerance ability. Therefore, the molecular evolution of the existing vacuolar Na~+/H~+ antiporter with higher salt tolerance ability and the cultivation of novel salt tolerant plants are significance for agricultural production.DNA family shuffling is a fast and powerful molecular biological tool for the directed evolution of enzymes, protein and antibody etc, which has a broad prospects for producing new enzymes with enhanced activity and stability, altered substrate specificity ect. In this study, DNA family shuffling was used to create chimeric Na~+/H~+ antiporters with improved transport activity. Two homologous Na~+/H~+ antiporters SeNHX1 and SsNHX1, from halophytes Salicornia europaea and Suaeda salsa, respectively, were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected.Yeast complementary test showed that SseNHX1 exhibited approximately1.4 and 1.2 times NaCl resistance in yeast growth than that of the two parental antiporters SsNHX1 and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 enabled yeast to accumulate more Na+ in the vacuole and increased its salt tolerance. Furthermore, this chimera also conferred increased tolerance to Li Cl and a similar tolerance to hygromycin B with the parental proteins in yeast, which suggested that the introduced mutations did not change the tolerance of Na~+/H~+ antiporter to other cation. Transient expression of the GFP-tagged SseNHX1 in onion epidermal cells showed that this novel antiporter still localized to the vacuolar membrane.To identify the physiological responses mediated by SseNHX1, transgenic tobacco plants overexpressing SseNHX1 and parental genes(SsNHX1, SeNHX1) were seperately generated with leaf disc via Agrobacterium-mediated transformation. Semi-quantitative RT-PCR showed that each of introduced NHX1 genes could be normally expressed. Salt resistance evaluation showed that transgenic plants overexprssing SseNHX1 grew faster, had significant higher root length, biomass and chlorophyll content compared to transgenic plants overexprssing parental genes and empty vector under 200 mM and 300 mM NaCl conditions. The SseNHX1 transgenic plants increased the net photosynthetic rate(Pn) and levels of proline, reduced accumulation of malondialdehyde(MDA) compared with other transgenic plants that separately transformed with SsNHX1, SeNHX1 and empty vector, accompanied by higher activities of antioxidant enzymes including superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT), and the greater accumulation of Na+ and K+ under 400 mM NaCl condition.