Functional Characterization Of The Nitrate Transporter GmNPF5.29 In Nodule Development And Symbiotic Nitrogen Fixation In Soybean

Nitrogen is an essential nutrient element for living organisms.In the soil,the nitrogen that plants can absorb and utilize is extremely limited,and the utilization rate of nitrogen fertilizer input is not optimistic.Therefore,nitrogen nutrition has become one of the important factors restricting plant growth.At the same time,nitrate（NO3-）,which is one of the forms of nitrogen absorption by plants,has also become a major research hotspot.The absorption of nitrate by plants mainly depends on the distribution of nitrate on the cell membrane with different affinities（low affinity（NRT1/NPF））And high affinity（NRT2）)transport system.Soybean（Glycine max）is one of the main crops in my country.Leguminous plants can specifically combine with Rhizobium to form nodule tissues and undergo symbiotic nitrogen fixation reactions.In the process of symbiotic nitrogen fixation,there are many transporters that play a certain role.The transcriptome data of many legumes indicate that NPF coding genes are strongly induced in root nodules,which indicates that NPF family genes are in the process of symbiotic nitrogen fixation in root nodules.Has played an indispensable role in legumes.So far,there have been few studies on NPF family genes in legumes.In our previous research,our laboratory discovered a NPF（GmNPF5.29）gene specifically expressed in root nodules.In order to further study whether this gene affects the growth and development of root nodules and the process of symbiotic nitrogen fixation,we designed a series of experiments to further explored the gene to clarify its gene function,in the hope that the study of this experiment will provide new ideas for the exploration of soybean nitrogen absorption and utilization through the nodule pathway.In this study,GmNPF5.29 was studied in depth through expression pattern analy sis,localization analysis,creation of mutants and overexpression materials.The results obtained are as follows:1.Through the NCBI website to search for its bioinformatics analysis,we learned that the gene is located on the eighth chromosome of the soybean genome.The full length of the ORF is 1758 bp,containing three introns and four exons,and the code is 585.A protein composed of three amino acids.2.We used a heterologous system to verify that the gene has the activity of a bsorbing NO₃^-under the conditions of p H 5.5 and 10m M ¹⁵NO₃^-.Through temporal and spatial expression patterns and localization analysis,we found that this gene is a gene expressed only in root nodules,mainly expressed in bacteroids,parenchyma cells and vascular bundles,and localized on the plasma membrane of the cell.3.The study on the mutant materials showed that the mutation of this gene has an inhibitory effect on the fresh weight,number,size of nitrogen concentration,a nd nitrogenase activity of plant nodules,while the activity of superoxide anion is in creased.There was no significant difference in the root phenotype.4.Research on overexpression materials showed that the overexpression of this gene had no effect on the growth and development of nodules and above-ground parts,and the fresh weight of roots increased significantly,but the overexpression of this gene significantly increased the nitrogen concentration of nodules and above-ground parts.There was no significant difference in nitrogen concentration in the roots.In summary,we found that the mutation of this gene severely hindered the growth and development of nodules and reduced the activity of nitrogenase.Therefore,we concluded that the gene GmNPF5.29 played an important role in the growth and development of nodules and symbiotic nitrogen fixation.This gene can affect the growth and development of nodules and the process of nitrogen fixation by affecting the nitrogen absorption and utilization of nodules.This result provides a new gene for the study of nutrient absorption and development of soybean nodules,and has important theoretical guiding significance.