Functional Characterization Of Overexpression For Nitrate Transporter Gene ZmNRT1.1B In Maize

Nitrogen(N)is one of the three important nutrient elements of plants and an important growth limiting factor.Appropriate application of nitrogen fertilizer in agricultural production can increase the photosynthetic efficiency of corn,thereby increasing yield.However,the nitrogen utilization rate of corn is low,only about 30%,so cultivating nitrogen efficient varieties is the key to improving nitrogen use efficiency.In this study,high-quality transformants were selected in the early stage for two-year field trials(Gongzhuling,Jilin),and three nitrogen fertilizer gradients(no nitrogen application,70% nitrogen application,and normal nitrogen application)were set up.The function of the ZmNRT1.1B gene was further identified through analysis of molecular evidence,agronomic traits,yield,and nitrogen content.In addition,RNA was extracted from the roots and leaves of the overexpression material and its isolated control seedling stage,and differential transcriptome analysis was performed to preliminarily analyze the possible genetic mechanism of the ZmNRT1.1B gene.After that,in order to explore the variation of the ZmNRT1.1B gene in natural populations,the polymorphism analysis of 60 maize inbred lines was carried out.It aims to provide a theoretical basis for cultivating nitrogen-efficient varieties and developing new molecular markers.The main findings are as follows:1.Through a two-year one-point field experiment,analyze the agronomic characteristics,total nitrogen content and yield of each transformation event.Compared with the control,each transformation event increased the yield of the crop by increasing the ear weight,ear length,ear row number,and 100-kernel weight of corn ears under low nitrogen and medium nitrogen conditions.2.Differential transcriptome analysis was performed on the overexpression material and the leaves and roots of the isolated control four-leaf one-heart stage.A total of 78.45 Gb Clean reads were obtained.The Clean reads in each library reached 6.20 Gb,and the Q30 was 92.29%and above,and The GC content is about 53%.About 87.26%-91.57% of the spliced sequences can be successfully aligned to the B73 v4 genome sequence.Using 7 major databases to annotate gene functions,a total of 4,396 new genes were discovered in leaves,of which 3305 genes were functionally annotated,and a total of 5925 new genes were discovered in roots,of which 4620 genes were functionally annotated.3.In this study,database mining and coding regions sequencing were used to analyze the polymorphisms of ZmNRT1.1B coding regions in 507 inbred lines from an association analysis genetic population and 60 maize inbred lines.The results showed that most of the mutant sites were identified in the 5’-UTR regions and intron regions of ZmNRT1.1B.The coding region of ZmNRT1.1B was highly conserved.Only 29 SNP sites and 1 In Del site were found in the coding sequence of 1788 bp,of which 23 synonymous mutations and 6 non-synonymous mutations were found.According to the sequence variation of the coding regions or the amino acid sequences translated from the coding regions with non-synonymous mutations and the In Del site,60 maize inbred lines were divided into 20 haplotypes or 8 haplotypes,respectively.The homologous modeling results showed that the mutation of Asp286 Ala in amino acid sequence and the insertion of Ala at 292 could lead to the change of protein folding and change the structure of the protein.