| Soybean,as a cereal crop with high protein and oil content,is the main source of plant-based protein and oil for humans.Soybeans originated in China,where historically they were the world’s leading producer,and currently China ranks fourth in soybean production worldwide.As demand and consumption of soybeans increase,so does production.In order to increase soybean production,the use of chemical fertilizers has also increased.Nitrogen contributes to crop yield by as much as 40-50%,with industrial nitrogen fertilizers being the most commonly used type.While heavy use of industrial nitrogen fertilizers has led to an increase in soybean production,it has also raised production costs and caused serious pollution problems such as soil compaction,eutrophication of water bodies,and excessive heavy metal contamination of soil.To overcome these problems,there are many research directions,but the most crucial one is to utilize symbiotic nitrogen fixation of legumes,which effectively alleviates the harm caused by chemical nitrogen fertilizers through the symbiosis between soybeans and rhizobia.There is a significant interaction between soil nitrogen content and rhizobia,and high soil nitrogen content directly suppresses nodule formation.Investigating the corresponding response of soybean nodulation traits to high and low nitrogen levels is of great significance for in-depth analysis of the molecular mechanism of symbiotic nitrogen fixation.These issues are still hot topics in the field of soybean-rhizobium symbiotic nitrogen fixation research and consequently,great deal of evidences have been emerged,which can help improve the nitrogen fixation efficiency of the legume-rhizobia symbiotic system,save production costs,increase production,and provide some theoretical support for genetic breeding of new soybean varieties with high nitrogen fixation efficiency.But the details on the molecular mechanisms have not yet been fully elucidated.This work focuses on the study of genes related to soybean and root nodulation.Firstly,from the soybean RIL line of our laboratory,QTLs related to root nodulation were identified and located,and 22 differentially expressed genes were then selected from the QTLs through transcriptome analysis.Gene monoallelic analysis was further performed to screen two candidate genes,including GmTCP（Glyma.19G095300）and GmNLP（Glyma.12G185700）,for the subsequent transgenic experiments.After that,Arabidopsis TCP and NLP mutants were complemented by transformatiton of GmTCP and GmNLP to investigate whether GmTCP and GmNLP regulate the root development of Arabidopsis under different nitrogen concentrations.Furthermore,the effects of overexpression or knockout of GmTCP and GmNLP on nodulation and root development in soybean using soybean hairy root transformation were investigated under different nitrogen concentrations.Lastly,the activity of the GmTCP promoter in rhizobia was analyzed.The details of the main research results are as follows:1.Mapping and mining of the genes with soybean response to nodulation:two QTL loci related to root nodules were identified and located through QTL analysis on chromosome 19 and chromosome 12.Transcriptome data further demonstrated that total 22 genes in these two QTL loci were differentially expressed to the high degrees during symbiotic nitrogen fixation.Single nucleotide polymorphism analysis for the 22 genes revealed that GmTCP and GmNLP have high level of the polymorphism in the DNA sequences among the parental lines of chromosome segment substitution series.The GmTCP and GmNLP were further investigated to analyze their effects on nodulation response to nitrogen concentration in soybeans.2.Investigating the effects of At TCP and At NLP mutations on root development in Arabidopsis:Arabidopsis homologous genes At TCP20（AT3G27010.1）and At NLP（AT2G27450.2）were obtained through bioinformatics analysis and then mutated.T2 and T3 generations of At TCP20 and At NLP mutants were screened and identified through PCR amplification and gel electrophoresis,and the root development under different nitrogen concentrations were analyzed.Under N starvation conditions,there was a significant difference in primary root length between the mutants and wild types.There was no significant difference in primary root length between the mutants and wild type at high nitrate concentration（5m M KNO3）,whereas significantly shorter for the mutants than wild types at low nitrate concentration（1m M KNO3）.The difference decreased with increasing nitrate concentration.These results indicate that At TCP20 and At NLP are important factors for regulating root development in response to nitrogen concentration.3.The effects of nitrogen concentration on root development in Arabidopsis GmTCP and GmNLP complementation lines:Expression vectors p SOY1-GmTCP and p SOY1-GmNLP were constructed and transformed into Arabidopsis At TCP and At NLP mutants through Agrobacterium-mediated transformation to complement the TCP and NLP mutations.The Arabidopsis complementation lines were screened and then used to analyze the effects of GmTCP and GmNLP on Arabidopsis root development at different nitrate concentrations.Complementation of GmTCP and GmNLP increased the primary root length at different nitrate concentrations.At high nitrate concentration（5 m M KNO3）,the primary root length was slightly longer compared to no or low nitrate concentration（1 m M KNO3）.These results indicate that the expression of GmTCP and GmNLP can complement the primary root growth in Arabidopsis At TCP20 and At NLP mutants at different nitrogen concentrations.4.GmTCP and GmNLP proteins subcellular localization and their effects on tobacco leaves:Gene expression profile data of Phytozome13.0 website revealed that GmTCP and GmNLP genes are specifically expressed in roots.Recombinant expression vectors p GWB5-GmTCP and p GWB5-GmNLP were constructed using gene recombination methods,and then transformed into tobacco leaf epidermal cells via Agrobacterium-mediated transformation and finally the subcellular localization status of GmTCP and GmNLP proteins was observed using fluorescence confocal microscopy.It was determined that GmTCP and GmNLP are located on the nucleus and cytoplasmic membrane of tobacco leaves.Additionally,trypan blue staining demonstrated that four-day long Agrobacterium-mediated transformation of GmTCP and GmNLP does not cause necrosis in tobacco leaves.5.The effects of nitrogen concentration-dependent responses of GmTCP and GmNLP on soybean nodulation:Overexpression and gene knockout of GmTCP and GmNLP were performed through hairy root transformation in soybeans and the effects of GmTCP and GmNLP on nodulation and root development under different nitrogen concentrations were then analyzed using the transgenic lines.In hairy roots transformed with an empty vector,at high nitrate concentration（5 m M KNO3）,the number and dry weight of nodules decreased significantly;while at low nitrate concentration（1 m M KNO3）,compared to the no-nitrogen condition,there was a slight decrease in the number of nodules but the effect was not significant.In hairy roots overexpressing GmTCP and GmNLP,the number and dry weight of nodules decreased as the nitrate concentration increased,and both were significantly lower compared to hairy roots transformed with an empty vector.In hairy roots with gene knockout of GmTCP and GmNLP,there was a slight decrease in the nodule phenotype as the nitrate concentration increased,but there was no significant difference between the root nodule phenotype of knockout hairy roots and those transformed with an empty vector under the N starvation conditions.Compared to hairy roots transformed with an empty vector,overexpression of GmTCP and GmNLP led to a decrease in the number and dry weight of nodules,while in knockout hairy roots,there was no difference or slightly higher nodule phenotype compared to hairy roots transformed with an empty vector.In summary,the genes GmTCP and GmNLP were demonstrated to be involved in regulating soybean nodulation in response to nitrogen concentration.6.Tissue-specific activity analysis for GmTCP promoter:GmTCP promoter region（3.0 kb）was cloned onto the p CAMBIA3301 vector which was then transformed into soybean hairy roots.The transformed soybean hairy roots were subjected to inoculation of Sinorhizobium fredii HH103,followed by anatomical microscopic observations of gus reporter gene expression in the hairy roots at different nitrogen concentrations and time points.The results showed that GmTCP was expressed during root development and nodule formation.In the late stage of nodulation,the GmTCPpro:GUS construct exhibited strong GUS expression throughout the nodules.Besides the nodule cells,GmTCP was also expressed in other root regions,especially in the root tip.Additionally,the higher the nitrate concentration,the higher the GUS expression.Overall,GmTCP was demonstrated as one of the key genes that are involved in regulating soybean nodulation in response to nitrate. |
