Effects Of OsPT2 Overexpression On The Nitrogen Fixation And Nitrogen Metabolism Of Transgenic Vegetable Soybean Under Phosphorus Deficiency

Vegetable soybean, also named as 'mao dou', is a specialty soybean [Glycine max (L.)Merrill] harvested as a fresh vegetable when the seeds are fully filled and the pods are still green. Symbiotic N2 fixation has high P requirements because it is an energy-dependent process. However, Pi availability is often limited due to slow diffusion and high fixation in soils, making P deficiency a major restriction for symbiotic nitrogen fixation and legume crop productivity. Therefore, developing legume varieties with effective N2 fixation under P-limited conditions will be important to reduce N and P fertilization and enhance agricultural sustainability. OsPT2, the rice phosphate transporter gene, is up-regulated by P deficiency in rice and functions in translocation of the stored Pi in the plant. Previously, we generated three T2 transgenic soybean plants expressing OsPT2 .gene which improved P accumulation. In the present study, whether OsPT2 overexpression enhances N2 fixation and ammonium assimilation were investigated under low-Pi conditions in pot culture in three T3 transgenic lines and wild type plants inoculated with Bradyrhizobium japonicum.This study will provided a basic foundation for development of novel vegetable soybean varieties, which have high N2 fixation capacity under P limitation, by genetic engineering.The main results are as follows:1. Three rhizobial strains (named as NAU1301, NAU1302 and NAU1303) isolated from fresh nodules of soybean plants. Three isolates were designated as Bradyrhizobium japonicum by colony characteristics and 16S-23S rDNA IGS gene sequencing. Nodulation test further confirmed that three isolates were rhizobia and the vegetable soybean plants inoculated with these Bradyrhizobium japonicum could form nodules on the roots, and have significantly greater plant fresh weight than the control plants receiving no Bradyrhizobium japonicum.2. Five levels of Pi supply (5, 10, 20, 40, and 1000 ?M) were applied to determine the effect of P deficiency on nodulation and nitrogen fixation. P deficiency significantly decreased the Pi and total P concentration in various plant organs. The Pi and total P concentration in various plant organs were significantly decreased under low Pi stress. P deficiency significantly inhibited the nodulation and N2 fixation of vegetable soybean plants, as evidenced by less plant height, leaf area, shoot mass, nodule mass nodule size and total N content than those in normal Pi condition. Under low Pi levels, nodule mass was more affected when compared with that of the shoot and root mass. qRT-PCR results showed that six genes were significantly decreased at several time points under low Pi condition compared with the normal Pi condition, including GmENOD40-1 GmENOD40-2,GmNIN1a, GmNIN1b, GmRICl and GmRIC2. The expression of LysM-type receptor kinase genes, GmNFR1?/?, GmNFR5?/?, GmSymRK?/?, were not affected by low Pi treatment in this 11-day time-course study. This results indicates that P deficiency did not affect the perception of NF signal and infection process, but affect the later steps of nodule development and growth by repressing the expression leves of some nodulatoin genes.3. T3 homozygous transgenic lines overexpressing OsPT2 were selected and subjected to determine the effects of OsPT2 overexpression on nodulation and nitrogen fixation under P deficiency in pot culture. The transgenic lines showed significantly better plant growth performance, seed weight and nodule development than the WT plants under low-Pi condition. Further, the P accumulation, total N and total ureide concentrations were significantly higher in the transgenic lines than in the wild type plants under low-Pi condition. qRT-PCR analysis showed that the expression levels of GmENOD40-1,GmENOD40-2 (two early nodulin genes), and GmLba (one leghemoglobin gene) were significantly increased in transgenic lines at 24 and 32 days after low-Pi treatment. The increased transcript levels of GmGS1?1 and GmGS1?2 (two cytosolic glutamine synthetase genes) in the transgenic lines were consistent with the increase in glutamine synthetase (GS,EC 6.3.1.2) activity at 32 days after low-Pi treatment. The increased transcript levels of GmLba in the transgenic lines were consistent with the increase in Lb concentration. These results indicated that the overexpression of OsPT2 in the three transgenic soybean lines improved P accumulation resulting in enhanced N2 fixation and ammonium assimilation under low-Pi conditions.