Cloning And Functional Analysis Of Nitrate Transporter Gene TaNRT2L12 In Wheat

Wheat(Triticum aestivum L.)is one of the most important crops in the world,and wheat production plays a pivotal role in China’s grain production.Nitrogen is one of the most necessary elements for plant growth and development,and improving nitrogen utilization efficiency can effectively increase wheat yield.Nitrate is the main form of nitrogen uptake from soil in wheat,and nitrate transport systems in higher plants are responsible for nitrate uptake and transport.Among them,nitrate transporter gene NRT2s(nitrate transporter 2)encode a class of high-affinity nitrate transporter proteins.In this paper,the Ta NRT2L12 s coding regions and promoters were cloned from wheat.Based on a panel of 32 common wheat accessions with high diversity,the sequence polymorphisms of in Ta NRT2L12 s in three genomes A,B and D were analyzed by sequencing.d CAPs markers were developed to distinguish gene haplotypes,and association analyses were performed to determine the superior alleles and haplotypes.Tissue and subcellular localization of the Ta NRT2L12-B were detected by q RT-PCR and transient expression.Transgenic Arabidopsis plants overexpressing Ta NRT2L12-B were observed to reveal the biological function.The results are as follows:(1)Ta NRT2L12-B is constitutively expressed in various organs of wheat,with the highest expression in root base.Its expression is up-regulated by low nitrogen and down-regulated by high nitrogen treatment.Ta NRT2L12-B located on the cell membrane,interacts with Ta NAR2.1 and Ta NAR2.2 in yeast.Overexpression of Ta NRT2L12-B in Arabidopsis inhibits the growth of main and lateral roots.(2)Polymorphism and functional marker development of Ta NRT2L12 s genomic coding region.The total length of the Ta NRT2L12-A/-B/-D coding region is 1521 bp,with only one exon and no intron.Thirty-eight SNPs were identified in the Ta NRT2L12-A coding region,11 SNPs in the Ta NRT2L12-B coding region,and no SNP in the Ta NRT2L12-D coding region in the panel of 32 wheat accessions.According to two SNPs in Ta NRT2L12-A coding region,the gene sequence in A genome is divided into three haplotypes.The marker A-CSNP2 is associated with plant height,length of penultimate internode and peduncle length,and A-CSNP2-A is the superior allele to reduce the plant height.According to two SNPs of the Ta NRT2L12-B,the B coding region is divided into three haplotypes.B-CSNP1-C and B-CSNP2-A are the superior alleles of high thousands-grain weight(TGW),and the Hap-6B-1 is a superior haplotype of TGW.(3)Polymorphism and functional marker development of Ta NRT2L12 s promoter region.The 2459 bp promoter region was amplified before the start codon of the Ta NRT2L12-B.Total of 25 SNPs and 1 In Del were detected in the Ta NRT2L12-B promoter region.The 2386 bp promoter region was amplified before the starting codon of the Ta NRT2L12-D,but there is no SNP identified in the promoter region.According to three SNPs,Ta NRT2L12-B promoter is divided into four haplotypes.The marker B-PSNP2 is significantly associated with spike length,and B-PSNP2-C is a superior allele to increase spike length.The marker B-PSNP3 is associated with spike length and seedling root angle,and B-PSNP3-A is a superior allele to increase spike length and root angle.