| Nitrogen(N)is one of the essential nutrients for plant growth and development.The N fertilizer provides an important guarantee for the improvement and stability of crop yield.However,excessive application of N fertilizer leads to resource waste,an increase in planting costs,and environmental pollution.One of the effective ways to solve the above problems is to identify the regulatory genes of N absorption and utilization,tap the genetic potential of N nutrition in crops,and breed N-efficient varieties.Rapeseed(Brassica napus L.)is one of the important oil crops in the world.Compared to other crops(such as cereals),rapeseed has higher N fertilizer requirements and lower N use efficiency.Our previous study found that the content of abscisic acid(ABA)in leaves of N-efficient rapeseed(48)was higher than that of N-inefficient rapeseed(44)after flowering.In this study,their response patterns to exogenous ABA were determined,and transcriptomics was used to identify genes related to N use efficiency(NUE)in rapeseed.The candidate genes involved in the N uptake and utilization were studied by molecular biology,physiological,and physiological methods,which provides a theoretical basis for breeding N-efficient rapeseed,and has important significance for N fertilizer reduction,and agricultural sustainable development.The following main research results were obtained:1.Rapeseed varieties with contrasting nitrogen use efficiency(NUE)and shoots and roots of rapeseed have different responses to exogenous ABA.In the pot experiment,exogenous ABA spraying on leaves could increase the number of grains per silique by promoting the redistribution of N from vegetative organs to grains,thus promoting the grain yield and N utilization efficiency of 48,while the opposite trend was observed in 44.Two genotypes of flowering rapeseed were treated with ABA under hydroponics and RNA-seq was performed.The results showed that the expression levels of several N metabolism genes(NRT2s,NAR2 s,NRs,NiR,GDHs,GSs)in the roots of 48 were higher than those of 44 under normal conditions,while the expression levels of most of these genes were lower than 44 after ABA treatment.2.The high-affinity nitrate transporter gene NRT2 s with its molecular companion NAR2 s and N metabolism enzyme coding genes(NRs,NiRs,GDHs,GSs)in rapeseed were identified as candidate genes related to NUE.KEGG functional enrichment analysis was performed for the differentially expressed genes between 48 and 44.Key signal pathways such as N metabolism,sulfur metabolism,ABC transporter,and MAPK signal transduction pathways were significantly enriched.The main genes involved in the N metabolism pathway are high-affinity transporters NRT2 s and its chaperone protein gene NAR2 s,nitrate reductase gene NRs,nitrite reductase gene NiRs,glutamate dehydrogenase gene GDHs,and glutamine synthetase gene GSs.In addition,WGCNA analysis identified two modules that were significantly related to the root of 48,among which the core genes mainly included the amino acid transporter gene AAPs,the core component of ABA signal transduction ABFs,and the ABA signal response gene NF-YC.3.Overexpression of BnaC8.NRT2.1b significantly increased the NUE of atnrt2.1plants and the N uptake efficiency(NUpE)of rapeseed plants,and the overexpression of BnaC2.NAR2.1 significantly increased the NUpE both in atnar2.1 and rapeseed plants.BnaC8.NRT2.1b and BnaC2.NAR2.1 were selected as the key candidate genes for functional study.The results showed that BnaC8.NRT2.1b was only expressed in the local root region,leaf edge,and stamen,while BnaC2.NAR2.1 was expressed in the whole root,leaf,and stamen.BnaC8.NRT2.1b and BnaC2.NAR2.1 are both membrane-localized proteins and interact with each other on the cell membrane.Overexpression of BnaC8.NRT2.1b and BnaC2.NAR2.1could complement the phenotype of Arabidopsis mutant seedlings to the wild-type level.Compared to wild-type plants,overexpression of BnaC8.NRT2.1b in atnrt2.1 mutant could increase single plant grain yield,whole plant N accumulation,NUpE,and NUE by 50%,41%,41%,and 50%,respectively,under low N conditions.Overexpression of BnaC2.NAR2.1 in atnar2.1 mutants can increase both plant N accumulation and NUpE by 32% under low N conditions.Similarly,overexpression of BnaC8.NRT2.1b and BnaC2.NAR2.1 can significantly increase the NUpE in rapeseed.4.BnaA5.AIB(BnaA05g01330D)is a negative regulator of N uptake and utilization in rapeseed.The leaves and roots of 48 and 44 at the flowering stage were treated with low N,and RNA-seq was performed.After a comprehensive analysis of the results of RNA-seq and BSA-seq obtained by our research group,47 genes were selected as the candidate genes.Further analysis revealed that one of the transcription factors,BnaA5.AIB,which was annotated as a b HLH-class transcription factor induced by ABA,was highly expressed in the root.The expression of BnaA5.AIB in the root of 48 was lower than that of 44,and there were 4 amino acid mutations between 48 and 44 in BnaA5.AIB.The primary root of the ataib mutant was significantly longer than that of the wild type under low N conditions,and the whole plant N accumulation was significantly higher than that of the wild type.GUS histochemical staining showed that BnaA5.AIB was mainly expressed in leaves,root tips,and flowers,and its encoded protein was located in the nucleus.Overexpression of BnaA5.AIB in Arabidopsis can significantly reduce NUpE and NUE,and overexpression of BnaA5.AIB can significantly reduce the NUpE in rapeseed.5.The molecular module ABA-BnaA5.AIB-BnaC2.NAR2.1 is involved in the regulation of N absorption in rapeseed.The results of double luciferase and yeast one hybridization showed that BnaA5.AIB could bind the CAATTG cis-element in the promoter region of BnaC2.NAR2.1 and negatively regulate the expression of BnaC2.NAR2.1.The expression of BnaA5.AIB was induced by ABA,while that of BnaC2.NAR2.1 was inhibited by ABA.In addition,the expression of BnaC2.NAR2.1 was decreased in BnaA5.AIB overexpression lines.These results identified that the ABA-BnaA5.AIB-BnaC2.NAR2.1 pathway was involved in the regulation of N uptake in rapeseed... |
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