| Vegetable soybean [Glycine max (L.) Merr.], also named as’mao dou’, it is rich in protein, amino acid, vitamins, minerals, fiber and so on, it has become highly popular with consumers. The production areas of vegetable soybean is continued to expand, owing to the increasing market demand. Vegetable soybean is produced mainly in the middle and lower reaches of the Yangtze River and southeast coastal areas of China with long history of cultivation. However, the yield and quality of vegetable soybean have been seriously affected by low concentration of phosphate in the red soil in southern region of China, due to the special physical and chemical properties of red soil, phosphorus can easily be fixed. OsPT6 is high-affinity phosphate transporter protein, plays a major role in the process of phosphate uptake and translocation throughout the plant. To improve the efficiency of phosphate acquisition and utilization of vegetable soybean, we introduce OsPT6 gene into vegetable soybean cultivar ’NY-1001’ through an Agrobacterium-mediated transformation method. Fertile transgenic vegetable soybean plants were generated successfully and the T2 homozygous transgenic lines were tested for the tolerance to low phosphorus stress. The main results are as follows:1. According to sequence of OsPT6 gene published in NCBI, we designed specific primers. OsPT6 gene coding region was introduced SacI and XbaI restriction site by PCR and then inserted between the CaMV35S promoter and the NOS terminator region of the modified vector pCAMBIA3301 to generate a pCAMBIA3301-OsPT6 by restriction endonuclease digestion and ligation. The plant expression vector was successful constructed with a bar gene as selection gene and an mtion-gus reporter gene encoding β-glucuronidase. Then the new construct was transformed into Agrobacterium tumefaciens stain EHA105, EHA105/pCAMBIA3301-OsPT6 was obtained and would be used for transformation.2. Two independent OsPTd-transgenic vegetable soybean plants (numbered 12PT6-1 and 12PT6-2) were successfully obtained using the Agrobacterium-mediated cotyledon-node transformation. Southern blot hybridization analysis revealed that the OsPT6 gene integrated into soybean genomes successfully with a single copy. The Basta painting showed that the To transgenic plants were conferred with herbicide-resistance. The T1 generation were analyzed by GUS assay and PCR analysis, and the results demonstrated that the exogenous genes were segregated in 3:1 ratio following a Mendelian segregation pattern. T2 progenies were screened for the presence of the transgenes and selected for transgene homozygosity. Three homozygous T2 transgenic lines 12PT6-1-1,12PT6-1-14 and 12PT6-2-8 were obtained. The overexpression of the OsPT6 gene of the T2 transgenic plants was detected by quantitative RT-PCR analysis.3. T2 homozygous transgenic lines were selected and subjected to testing for phosphate acquisition efficiency by hydroponic culture. The Pi and total P contents in the leaves, stems and roots of the transgenic plants were significantly higher than those of the non-transformed (NT) plants under the low-Pi (10 μM Pi) and normal-Pi (1000μM Pi) condition, respectively. Under low-Pi stress, the transgenic plants grew better and exhibited significant increases in plant height, root length, root weight, the leaf area, and the yield compared with the NT plants. Our data indicate that the overexpression of OsPT6 in transgenic vegetable soybean lines improves of phosphate acquisition efficiency. |
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