| Wheat is an important food crop in the world.Nitrogen,as an important inorganic nutrient,plays an important role in regulating various physiological processes such as wheat growth and development and crop yield formation.Therefore,improving the Nitrogen Use Efficiency(NUE)of wheat plays an important role in the sustainable development of agriculture.NRT2(Nitrate Transporter 2)and the transcription factor TCP are important factors in plant nitrogen utilization,but the systematic studies on NRT2 and TCP in wheat are not yet comprehensive.In this paper,through the systematic analysis of wheat NRT2 and TCP,the NRT2 and TCP genes that respond to nitrogen starvation in wheat were screened out,and the gene function was preliminarily analyzed to provide candidate gene resources for the screening and utilization of wheat nitrogen-efficient germplasm,and at the same time for wheat nitrogen use mechanism research to lay a theoretical foundation.The main research results obtained are as follows:1.Using bioinformatics analysis,46 NRT2 genes were identified in the whole wheat genome.The results of phylogenetic analysis showed that the family was divided into 3 subfamilies;gene structure analysis found that only 4 NRT2 genes each had an intron;the concatenated repeat analysis showed that TaNRT2.4.6-D has a pair of tandemly duplicated genes;promoter cis-acting element analysis showed that most family members of TaNRT2 have hormone stress response elements,of which 43 genes contained Me JA response elements CGTCA-motif in their promoters;gene expression pattern analysis under nitrogen starvation conditions found: the expression levels of TaNRT2.1.1-A,TaNRT2.1.3-A/B,TaNRT2.1.4-B,TaNRT2.1.5-B,TaNRT2.2-B,TaNRT2.4.1-A,TaNRT2.4.2-B/D,TaNRT2.4.3-B,TaNRT2.4.6-B and TaNRT2.7-B/D were significantly increased under nitrogen starvation conditions.Further gene function analysis found that TaNRT2.1.1-A,TaNRT2.1.3-A/B,TaNRT2.2-B,TaNRT2.4.1-A and TaNRT2.4.2-B genes were higher expressed in lownitrogen and high-efficiency wheat varieties than in low-nitrogen and low-efficiency varieties.2.TaNRT2.1.3-B gene was cloned from wheat and its encoded protein was analyzed by bioinformatics.The results showed that the coding region of TaNRT2.1.3-B gene was 1698 bp long and encoded a protein with 565 amino acids.Further prediction analysis showed that TaNRT2.1.3-B protein contained a conserved MFS＿1 domain,which was located in the cytoplasmic membrane.The evolution analysis of NRT2.1 protein among different species showed that TaNRT2.1.3-B was the closest relative to the homologous protein in durum wheat(Triticum turgidum L.).The expression patterns of TaNRT2.1.3-B gene in different tissues of seedling stage and under nitrogen starvation conditions were analyzed by real-time quantitative PCR.The results showed that the expression level of TaNRT2.1.3-B in roots was much higher than that in shoots;Under nitrogen starvation conditions,the expression level in low nitrogen and high efficiency wheat varieties was significantly higher than that in low nitrogen and low efficiency wheat varieties.3.In the whole wheat genome,a total of 60 TCP genes were identified by bioinformatics analysis.Phylogenetic analysis of the family was divided into three subfamilies;gene structure analysis found that 34 genes had no introns;most members of TaTCPs contained hormone stress response elements ABRE,TGACG-motif and CGTCA-motif,of which TaPCF6-D had up to 9 ABREs,TaPCF2-A and TaPCF8-D contain 6 ABREs respectively,TaTCP21-D contains 6 TGACG-motifs.Tissue-specific expression analysis showed that 2 genes TaTCP19-B/D were specifically expressed in roots,3 The genes TaTCP17-A/B/D were specifically expressed in panicle.The gene expression pattern analysis showed that the expression levels of TaPCF5-A/B/D and TaTCP19-D were significantly higher in low-nitrogen and high-efficiency cultivar compared with the high-nitrogen-high-efficient cultivar;and TaPCF5-B and TaTCP19-D had a higher expression level in the low-nitrogen and high-efficiency cultivar Kenong 199 in either normal nitrogen concentration environment or nitrogen starvation environment.Gene TaTCP19-B responds strongly to nitrogen starvation signal in high-nitrogen and high-efficiency cultivar AK58.Using the method of homologous gene cloning,the homologous gene TaTCP19-B of rice nitrogen efficient gene Os TCP19 in wheat was found,and the function of this gene was studied in depth.Using the yeast library,the mating method was used to screen the interacting proteins of TaTCP19-B,and two homologous genes TaTLP-4A/4B that encoding thaumatin-like proteins,were preliminarily screened. |
