| Nitrogen(N)is the most essential macronutrients for higher plants,which is not only important for plant growth and development,but also is crucial for crop yield and quality.Brassica napus is the most important oil crop in China.The growth,development,as well as production of B.napus need a large demand for N fertilizer.Therefore,in order to maintain or increase the yield of B.napus,N fertilizer was excessively applied in the yield.In fact,the N utilization efficiency of B.napus is low,which caused lots of problems,such as restricting the increasement of yield,wasting N fertilizer resource,and leading to environmental degradation.Thus,low N utilization efficiency is a prominent problem that restricts the development of B.napus production in China.Accordingly,improving the N utilization efficiency of B.napus is an important scientific issue that should be solved urgently in B.napus production in our country,and it is also an important way to ensure the stable and high yield of B.napus,to improve the quality and efficiency of B.napus production,and to promote sustainable agricultural development.In the past three decades,the screening and mechanism study of the functional genes involved in the N uptake,transport and assimilation gene networks has been an international research hotspot.To date,the gene networks related to N utilization have been well understood in model plant,Arabidopsis,however,there is still a lack of systematic research on the related gene networks in most plants,especially in crops,such as B.napus.In the present study,we mainly performed three aspects of research works:Firstly,we constructed the gene network of the N utilization pathway in B.napus genome,and then carried out a systematic bioinformatics analysis(such as subcellular location,chromosome location,gene duplication/expansion mechanism,and transcriptional regulating mechanism,etc);Secondly,we analyzed the spatio-temporal and low N stress expression profiles of the N utilization pathway in B.napus respectively based on two RNA-seq datasets,constructed its co-expression gene network,and then screened the differentially expressed genes(DEGs)and candidate genes that may be involved in high N utilization efficiency this pathway.Thirdly,we systematacially analyzed the origin,distribution,gene expansion and molecular evolution mechanisms of the N utilization pathway across 22 representative plant species at genome-wide level(including chlorophytes,mosses,gymnosperms and angiosperms).Together,the results of this study provide important gene resource for further study on the molecular mechanism of the candidates in B.napus N utilization efficiency,and also provide a new strategy for molecular breeding through promoting N utilization efficiency.The main results of this study are as follows:1.Genome-wide analysis of the N utilization related genes in B.napusIn this study,we identified 605 genes of the N uptake(201 genes),N transport(206genes)and N assimilation(56 genes)networks,and the common regulating genes of N uptake and assimilation networks(142 genes)in B.napus.The subcellular localization analysis showed that the majority regulating proteins(>90%)of the N utilization pathway were located in nucleus,while the structural proteins in this pathway were mainly located in cell membrane,vacuole membrane and plastids.Chromosome localization analysis showed that the N utilization related genes were unequal distributed on the 19 chromosomes of B.napus,and their distribution trends between A_n-and C_n-subgenomes were similar with no obvious bias.Collinearity analysis showed that the allopolyploid(273 genes,～58.5%)between its ancestor Brassica rapa and Brassica oleracea was the main driving force for large gene expansion of the N utilization pathway in B.napus.Meanwhile,small-scale duplication events also play important roles in the rapid gene expansion of this pathway in B.napus,including segmental exchange(80 genes,～17.1%),segmental duplication(67 genes,～14.3%)and homologous exchange(47 genes,～10.1%)events.Notably,the genes derived from B.rapa genome were tended to be duplicated and retained in B.napus.Cis-acting element and transcription factor binding site prediction showed that excepting the common core cis-element and lots of light responsive cis-elements,many hormone responsive cis-elements,abiotic stress responsive cis-elements and transcription factor(TF)binding sites were widely observed in the promoter regions of the N utilization related genes,suggesting the N utilization pathway may be influenced by the corresponding factors.Mi RNA regulatory site prediction showed that 37 types of mi RNAs may have156 potential regulatory sites in nitrogen utilization related genes,suggesting the potential roles of mi RNAs in regulating N utilization pathway.2.Expression profile analyses of N utilization related genes and screening candidate genesWe further analyzed the spatio-temporal and low N stress expression profiles of the N utilization related genes based on the RNA-seq datasets constructed by our laboratory.The spatio-temporal expression profile analysis showed that 527 of 605 genes of the N utilization pathway had detectable expression levels(FPKM≥1)in the 60 samples of 7major organs at different development stages in B.napus.Among the 527 genes,454(～86.1%)and 393(～74.6%)genes were preferential expressed in roots and leaves,respectively.Moreover,49 of 114 duplicated pairs of the N utilization pathway exhibited different expression patterns(Pearson correlation coefficient<0.6),suggesting their functional divergent during the evolution.Low N stress expression profile analysis showed that 463 of 605 N utilization related genes(～76.53%)had detectable expression levels(FPKM≥1)in roots and/or leaves,and 17 of them were specifically expressed under low N stress(they were not expressed under normal condition).The largest number of DEGs of the N utilization pathway was after 12 days under low N treatment,including 203 and 166 genes were differentially expressed in roots and leaves respectively.Based on the results of spatio-temporal and low N stress expression profiles analysis as well as co-expression analyses,we screened 17 candidate genes(e.g.Bna NRT2.3,Bna NPF7.3 and Bna TGA1.2,etc.)that had high expression levels and also had obvious inducible expression profiles under low N stress.3.The origin and evolution mechanisms of N utilization gene network in plant kingdomIn this study,we obtained 4262 candidates from 27 gene families that are involved in N utilization pathway(including N uptake,transport and assimilation networks)in 22representative plant genomes by Blast P method.We found that the structural genes of the N utilization pathway have already existed in aquatic plants(Chlamydomonas reinhardtii and Volvox carteri),whereas the TFs that were involved in regulating N uptake and assimilation processes were mainly arisen from lower land plants,Marchantia polymorpha and Physcomitrella patens,and the major TFs in this pathway were firstly emerged in early angiosperm Amborella trichopoda.These results indicated that the gene regulatory network of the N utilization pathway was mainly originated in early angiosperm,and then become more complex along with the evolution.Moreover,we found that number of the N utilization related genes in higher plant genomes was obviously larger than that of lower plants,indicating that the related genes had underwent significant expansion during the evolution.The number of most structural genes in the N utilization pathway begin to expand in algae,and then it was stable in monocot and dicot plants.However,the number of most TFs has been expanding along with plant evolution.These results indicated that the structural gene network of the N utilization pathway had been perfected in higher plants,while its regulating gene network has been evolving in higher plants,implying that the regulating genes are responsible for the different N utilization efficiency of different plants/crops.In addition,selective pressure analysis showed that most genes in the N utilization pathway were under purifying selection,except that members of 3 gene families have significant positive selection sites,including b ZIP1s(7 sites),HRS1/HHO1s(25 sites)and GSs(54sites).This suggested that purifying selection is the main driving force of the N utilization pathway during the evolution.The comparative expression profile analysis of the N utilization related genes in five typical land plants(M.polymorpha,Oryza sativa,Zea mays,B.oleracea,and Arabidopsis)indicates that the expression profiles of N utilization related genes were relatively conserved among different species,implying their functions were conserved during the evolution. |
