Genetic Transformation Of StNHX1 And OsPT2 Genes To Produce New Germplasms Enhancing To Salt And Phosphorus Utilization Efficiency In Vegetable Soybean

Vegetable soybean [Glycine max (L.) Merrill], a legume-vegetable rich in proteins, minerals, and various vitamins that possesses a specific flavor and taste, is highly popular in China, particularly in the southern region of the Yangtze River. With the expansion of market demand, the cultivation area of vegetable soybean is increasing rapidly. However, the yield and quality of vegetable soybean have been seriously affected by the constant exacerbation of soil salinization and low concentrations of phosphates (low-Pi). In this study, we have transferred the wild eggplant’Torvum Vigor’(Solanum torvum Swartz) vacuolar Na+/H+antiporter gene(StNHXl) and rice (Oryza sativa L.) phosphate transporter gene (OsPT2) into vegetable soybean cv.’Xinliaoxian’, and tested the tolerance to salt and low-Pi stress of their progenies, respectively. The main results were as follows:1. A novel vacuolar Na+/H+ antiporter gene from wild eggplant’Torvum Vigor’ (StNHX1; Accession No.:JN606860.1) was isolated. Sequence analysis showed that the ORF of StNHX1 gene is 1,599 bp, encoding 532 amino acids. Characteristic analysis indicated that StNHXl shared high nucleotide sequences identity with the SINHX1 from Solanum lycopersicum. The unrooted phylogenetic tree of StNHX1 and other species suggested that StNHX1 had the closest relationship with tomato and the furthest relationship with Salicornia europaea.2. The target gene (StNHX1 or OsPT2) was directional inserted into the plant binary vector pCMBIA3301 using restriction sites (BamHI and SacI or SacI and XbaI). The plant transformation vectors pCMBIA3301-StNHX1 and pCMBIA3301-OsPr2 were successful constructed, in which the target genes were promoted by the CaMV35S promoter and the gus and bar genes were used for reporter gene and selection gene, respectively. Then the new constructs were transformed into Agrobacterium tumefaciens stain EHA105, respectively, which would be used for transformation.3. The vacuolar Na+/H+ antiporter gene from ’Torvum Vigor’, StNHX1, was transferred into the vegetable soybean cultivax ’Xinliaoxian’ through an Agrobacterium-mediated protocol. PCR and Southern blot analyses confirmed the successful integration of the exogenous genes. The Basta(?) painting showed that the transgenic plants were conferred with herbicide-resistance. GUS assay and semi-quantitative RT-PCR analysis demonstrated that the alien genes were inherited by the progenies and that the StNHXl gene was overexpressed in the T3 generation. The overexpression of this gene conferred high tolerance to salt stress. Under NaCl stress, the scorch scores and the Na+ and malondialdehyde (MDA) contents of the leaves of the transgenic lines were significantly lower than those of the wild-type (WT) plants, whereas the K+/Na+ ratios, the contents of K+, relative chlorophyll, and relative water, and some morphological traits of the transgenic lines were significantly higher compared with those of the WT plants. These results indicate that the overexpression of StNHXl enhances the salt tolerance of vegetable soybean and that StNHXl is one of the promising target genes that can be manipulated to improve the salinity tolerance of crops.4. Cotyledonary-node explants of the vegetable soybean were inoculated with the Agrobacterium tumefaciens strain EHA105 harboring the vector pCAMBIA3301-OsT2, which contained OsPT2, gus and bar genes. The Basta(?) painting showed that the transgenic plants were conferred with herbicide-resistance. Ten fertile To transgenic plants were obtained and semi-quantitative RT-PCR of progenies demonstrated that OsPT2 gene was overexpressing in the T2 generation. Three T2 transgenic lines overexpressing OsPT2 were selected and subjected to testing for tolerance to low concentrations of Pi (low-Pi; 20 μM Pi) by hydroponic culture using modified Hoagland’s nutrient solution. The total P contents in the leaves, stems, roots, and seeds of the transgenic plants were significantly increased under the concentrations of low-Pi and 1000 μM Pi of standard Hoagland’s nutrient solution, respectively. Under low-Pi stress, the yields of the transgenic lines were significantly higher than those of the WT plants. Taken together our data suggest that the overexpression of OsPT2 in transgenic soybean lines improves Pi acquisition and seed yield, and OsPT2 may serve as one of the promising target genes that can be manipulated in crop improvement for minor use of Pi fertilizers.In summary, our data suggest that StNHXl transgenic vegetable soybean has a strong tolerance to salt stress and OsPT2 transgenic vegetable soybean has more efficiency of Pi acquisition and utilization, and StNHX1 and OsPT2 may serve as the promising target genes that can be manipulated in crop improvement for cultivation in saline and low-Pi soils, respectively.