Salt Tolerance Analysis Of TaNHX2 Over-expression Transgenic Soybean And Preliminary Phenotype Identification Of NTHK1 Over-expression Transgenic Soybean

Soil alkalization and secondary salinization have become a global ecological and resources issue. Soybean is an important grain and oil crop, as a moderate salt-tolerant crop, its growth and development severely affected by salt stress, thus leads to substantial decreasing in yield. Using the transgenic technology to create new soybean varieties is one of the important methods to solve the soil salinity problem. So far, researchers have got some new salt-tolerant soybean germplasm through genetic engineering, while these studies only infer to preliminary identification of early generation of transgenic soybean lines but fail to analyze the yield performance of transgenic lines under salt stress and far from applying in soybean production.We chose the T4 generation of TaNHX2(wheat Na+/H+ transporter code gene) over-expression transgenic soybeans, which the transgenic authenticity confirmed by Glufosinate-resistance assay, PCR and RT-PCR detections at V3 stage. The positive individuals were then treated with NaCl solution at V4 stage, during the treatment phase, the salt induced phenotype of transgenic and wild-type plants was observed. Furthermore, we measured the Photosynthesis intensity of the soybeans at R3 stage. Finally, the plant height, node number on main stem, pod number per plant, seed number per plant and seed weight per plant of soybean plants which harvested at R8 stage were measured. Two concentrations of NaCl solution(0mM and 200mM) were applied for the treatment in Beipuchang research site, while three concentrations were applied(0mM, 150 mM and 200mM) in Changping, Beijing. The observation of salt induced phenotype and measurement of Photosynthesis intensity only taken in Beipuchang research site. The successful expression of foreign gene TaNHX2 was confirmed by molecular identification, its inheritance is stable. When compare with wild-type, transgenic soybean lines(C12, C21 and C19) had lower salt damage degree and fewer negative effects on photosynthesis under 200 mM NaCl treatment, the transgenic lines kept in a higher photosynthesis compare to the wild type. In addition, the decreasing rate of transgenic soybean lines’ yield are less than wild-type under 150 mM and 200 mM NaCl stress, the plant height, pod number per plant, seed number per plant and seed weight per plant of lines C12, C21 and C19 were obviously higher than wild-type, line C19 showed an outstanding yield potential both in two experimental research sites. Therefore, it is practical valuable for breeding and production.In addition, in order to explore the functional role of tobacco ethylene receptor encoding gene NTHK1 in soybean salt stress response, we chose the T4 generation of NTHK1 over-expression transgenic soybean which confirmed by Glufosinate-resistance assay, PCR and RT-PCR detections. Subsequently, the samples were taken from the positive plants under NaCl stress, measured the relative expression level of NTHK1 and some salt responsive transcriptional factors encoding genes. Meanwhile, in order to detect the ethylene sensitivity of transgenic soybean lines, we sowed the seeds on B5 medium, containing different concentrations of ethylene precursor ACC(1-aminocyclopropane-1-carboxylic acid), and cultivated at dark. Finally, we identified the salt-tolerance level of transgenic soybeans under hydroponic conditions treated with NaCl. The results showed that the expression of the foreign gene NTHK1 in transgenic lines was induced by salt stress. The salt responsive elements of NTHK1 may locate in its coding region. Furthermore, the expression level of GmDREB2A、GmDREB3、GmNAC3 and GmNAC20 are higher in transgenic lines than wild-type, indicating that NTHK1 may involve in regulation of these genes, especially GmDREB3. When comparing with wild-type, the ethylene sensitivity of transgenic soybean is decreasing while the salt sensitivity is increasing. Combing our results with previous findings, we speculated that NTHK1 may involve in plant salt sensitive response or decrease the salt tolerance of plant by negative regulation of ethylene response.