Functional Analysis Of GmNRT1.2 Regulating Nodule Development Of Soybean

Nitrogen is one of necessary elements for plant growth and development.Plant roots can obtain nitrate nitrogen and ammonium nitrogen from the soil.Nitrate transporter(NRT)is mainly responsible for the absorption of nitrate.Especially,legumes can also initiate specific symbiotic nitrogen fixation systems to obtain nitrogen nutrition.However,the NRT family protein involved in the nitrogen uptake and symbiotic nitrogen fixation process of soybean is not reported.In this study,we investigated the effects of different nitrate concentrations on the growth and development of soybean.Moreover,the function and molecular mechanism of the homologous gene NRT1.2 in nitrogen absorption and symbiotic nitrogen fixation were conducted research.The following research results were obtained:1.The effects of different nitrate concentrations on soybean growth and development were studied.With the increasing of nitrate concentration,the primary root length,the number of lateral roots,the total length of all lateral roots,and the fresh weight of roots were clearly changed.At the same time,the nitrogen content in different tissues was dramatically increased.Under the same concentration of external nitrate,the nitrogen content in soybean roots,stems,cotyledons and leaves showed to be increased significantly.In addition,with the increasing of nitrate concentration,the nodule numbers was found to be decreased significantly in soybean.2.Six homologous genes of At NRT1.2 obtained in soybean were analyzed by different bioinformatic analysis.It was found that GmNRT1.2a,GmNRT1.2b,GmNRT1.2c and GmNRT1.2d is far away from GmNRT1.2e and GmNRT1.2f through phylogenetic analysis.Based on the ratio of d N/d S between GmNRT1.2a and GmNRT1.2b,it was showed that some sense mutations were generated during evolution.Based on the expression data collected from Soybean e FP and Soy Base,GmNRT1.2a and GmNRT1.2b appeared to be highly expressed in roots and/or nodules.These results suggest that there may be functional differentiation of GmNRT1.2s in soybean.3.The expression pattern of candidate genes in different tissues and in response to nitrogen or Nod Factor treatment were analyzed by RT-qPCR.Based on these results,GmNRT1.2a,GmNRT1.2b and GmNRT1.2c showed to be significantly expressed in leaves and roots.Interestingly,after nitrate treatment,GmNRT1.2a and GmNRT1.2b were found to be significantly induced by nitrate.In addition,the expression of GmNRT1.2a and GmNRT1.2b were promoted at 3 days after Nod Factor inoculation.However,GmNRT1.2c did not induce by Nod Factor.4.Using soybean hairy root transformation system,GmNRT1.2a and GmNRT1.2b were found to promote nodulation of soybean.Overexpression GmNRT1.2a or GmNRT1.2b at low nitrate concentrations could clearly increase the number of nodules.However,the nodule numbers of transgenic hairy roots having GmNRT1.2a and GmNRT1.2b overexpression did not affect the inhibition of nodulation under the condition of high nitrate concentration.5.Using tobacco transient transformation system,GmNRT1.2a and GmNRT1.2b might be located on the plasma membrane.In addition,GmNRT1.2a might interact with Gm NFR5α in bimolecular fluorescence complementation system(Bi FC).In summary,the growth and development of soybean was significantly affected by the nitrate concentration.GmNRT1.2a and GmNRT1.2b might regulate the nodule numbers through Nod Factor signaling pathway.Our results provide some clues for uncovering the molecular mechanisms modulating nitrogen absorption and symbiotic nitrogen fixation in soybean.