Molecular Cloning, Functional Analysis And Genetic Transformation Of A Na~+/H~+ Antiporter Gene GmNHX1 From Soybean

Soybean (Glycine max) is an major grain and oil crops, and the main source of edible oil and vegetable protein of the world. In order to meet the development requirements of modern agriculture, soybean resistance enhancement is one of the development direction of soybean breeding. With the development of molecular biology and genetic engineering technology, transferring salt tolerance genes into high-quality soybean genomes is become an effective way to improve soybean salt tolerance. Na+/H+ antiporter protein could export cytosolic Na+ outside the cell or separate Na+ in the vacuole so that to maintain the plant cytoplasm Na+ stability, Na+/K+ relatively ratio, osmotic pressure, and reduce salt stress. In this study, we have cloned the salt tolerance gene GmNHX1 from soybean varieties Jidou 7 and transferred it into Arabidopsis to test gene function on salt tolerance; we further optimized soybean embryonic tips regeneration and transformation system based on laboratory previous work; we transferred GmNHX1 into salt-sensitive soybean varieties with Agrobacterium-mediated transformation method. The results show that:1. We have cloned Na+/H+ antiporter gene GmNHX1 from soybean varieties Jidou 7 by RT-PCR. Bioinformatics analysis indicated GmNHX1 is highly homologous with the function known tonoplast Na+/H+ antiporter. Semi-quantitative RT-PCR analysis indicated that GmNHX1 expression is constitutive and increased under NaCl stress. Under salt stress the GmNHX1 expression level in leaves was significantly higher than that in root. Salt-tolerant varieties Jidou 7 has a lower GmNHX1 background expression level than salt-sensitive soybean varieties Jidou 17, but a higher increase under salt stress. And this could afford a primary evidence that GmNHX1 have a relationship with soybean response to solt strees.2. The binary expression vector pCAMBIA1300-35S::GmNHX1-eYFP was obtained and then transformed into the Arabidopsis theliana. The salt tolerance phenotype in Arabidopsis GmNHX1 overexpression lines under 150mM NaCl indicated GmNHX1 could improve salt tolerance in plants.3. We optimizated the soybean embryonic tip regeneration system. The results show that under 0.2~0.5 mg/L 6-BA the four soybean genotypes had better embryonic tip regeneration rates and regeneration rate of Jidou 16 is the best. Under 0.2~1.0 mg/L TDZ the four soybean genotypes had better embryonic tip regeneration rates; nf37 has a highest shooting rate and plant regeneration rate.4. Determined the optimal Basta screening concentration to the tested soybean varieties embryonic tip regenerated seedlings was 0.6 mg/L; but to the seed germinated seedlings the optimal Basta screening concentration was 200 mg/L.5. We obtained the trans-GmNHX1 gene PCR positive soybean plants by Agrobacterium-mediated embryonic tips genetic transformation using the optimized embryonic tips regeneration system.