Functional Analysis And Soybean Genetic Transformation Of A Na~+/H~+ Antiporter Gene Gm NHX1

Soybean [Glycine max(L.) Merrill] is an important food and oil crop. However, soil salinization has become one of the major limiting factors affecting soybean growth and yield. Genetic engineering may offer a new solution to solve this major limitation. Transgenetic operation have been performed carring stress related genes into fine varieties to induce its stress related performance. Multiple gene that are related to salt tolerance bave been applied in plant stress tolerance research, members in Na+/H+ antiporter family have been studied in-depth. Na+/H+ antiporter functions in cytoplasm through Na+ efflux or Na+ compartmentation into the vacuole to maintain Na+ homeostasis and Na+/K+ relatively stable cytoplasmic ratio, reducing salt stress on plant damage. However, little is known about the soybean Na+/H+ antiporter gene and its potential application for salt tolerance. Here we report overexpression of Gm NHX1 genes in Arabidopsis and yeast nhx1 deletion mutant. We employed NMT technology, real-time PCR and yeast complementation tests to study the Gm NHX1 functions and it role in salt tolerance. Using particle bombardment-mediated transformation of onion, we observed sub cellular localization of Gm NHX1 protein. Then, we overexpressed this Gm NHX1 in transgenic soybean derived from Agrobacterium tumefaciens-mediated transformation. The results are summarized as follows:1. Identification of Arabidopsis T3 homozygous lines overexpressing Gm NHX1. Three homozygote lines were identified through antibiotics screen and Southern blot analysis and all three lines were found to carry single copy Gm NHX1 transgened. RT-PCR analysis showed that the overexpress of Gm NHX1 was much higher in leaves than in roots.2. Identification of Gm NHX1 overexpression lines displaying salt tolerance. We tested salt tolerance of Gm NHX1 overexpression lines during seed germination stage. Under 0-150 mmol/L Na Cl treatment condition, all three transgenic lines had longer taproot than wild-type and growed healthier; after seed germination, line OE1-1 grow healthier than the wild-type, and have longer taproots under 150 mmol/L Na Cl salt stress condition; In developed plants, all three lines growed better than the wild type, and had more dry and fresh weights, more number of rosette leaves and plant height under 170 mmol/L Na Cl treatment for three weeks after seed germination. Thus, we concluded that transgenic Arabidopsis overexpressing Gm NHX1 is more salt tolerant than WT plants.3. Construction of a yeast expression vector p YES2-Gm NHX1 and functional complementation through nhx1 salt-sensitive yeast mutant strains YDR456 W and AXT3. These tests were conducted under 50 or 200 mmol/L Na Cl treatment condition. The results showed that yeast mutant strains overexpressing transgenic Gm NHX1 gene growed faster than the wild type, and it is possible to restore the salt tolerance of yeast mutants.4. Construction p CAMBIA1300-35S::Gm NHX1-GFP vector for subcellular localization. Using Biolistic-mediated transformation of onion epidermal cells and fluorescence microscopy, we found that the Gm NHX1-GFP was mainly distributed in the vacuole membrane.5. Salt tolerance mechanisms of overexpressing soybean Gm NHX1 gene. After 170 mmol/L Na Cl treatment for two weeks, transgenic Arabidopsis leaves showed much higher K+ and Na+ contents than WT; in roots, the transgenic plants showed increased K+, but not Na+ content. NMT technology(NMT) results showed that after salt treatment the Na+ efflux rate of OE1-1 was significantly greater than WT. Using BCECF-AM dye-labeled H+, fluorescence microscopy were used to observe chances of H+ content in developing roots, which showed a high p H value matained in OE1-1 plants. We also examined the transcript profiles of other salt-tolerance related genes and showed that after salt treatment the over expression Gm NHX1 mainly affects At SKOR and At SOS1 gene expression.6. We employed Agrobacterium-mediated transformation of soybean cotyledon node system to introduce Gm NHX1 genes into salt-sensitive soybean genotype Wuxing 2 and confirmed 15 T0 transgenic events by herbicide screen and PCR.