Cloning And Functional Analysis Of Spinach Nitrate Transporter Gene

Nitrate nitrogen?NO₃^--N?is one of the most important nitrogen nutrients in spinach,which is closely related to the growth and development,morphogenesis and yield quality of spinach.Nitrate transporters are involved in the active uptake NO₃^--N in plants,which play an important role in root absorption and vivo distribution of nitrate nitrogen.Studying spinach nitrate transporter and its biological functions are helpful for improving the absorption and transport capacity of nitrate,reducing nitrate accumulation and increasing nitrogen use efficiency in spinach.In this paper,we cloned five spinach nitrate transporter genes?SoNRT1.3,SoNRT1.5,SoNRT1.9,SoNRT2 and SoNRT3?from spinach material SP4.The bioinformatic analysis and expression patterns of these genes each gene under different nitrate concentration treatments were studied.Moreover,the possible biological functions of three nitrate transporters were also studied in Arabidopsis thaliana.The main results are as follows:1.Sequence analysis showed that SoNRT1.3,SoNRT1.5,SoNRT1.9 and SoNRT2genes contain MFS conserved sequences,and their amino acid sequences have higher homology with their Arabidopsis thaliana orthologs.The secondary structures of SoNRT1.3,SoNRT1.5,SoNRT1.9,SoNRT2 and SoNRT3 are mainly composed of?-helix and irregular curl.They contain 12 transmembrane domains except of SoNRT3.Real-time quantitative PCR analysis showed that SoNRT1.3 and SoNRT3 were mainly expressed in roots,while SoNRT1.5,SoNRT1.9 and SoNRT2 had considerable expression levels both in roots and shoots.Both nitrogen deficiency and nitrogen supply induced the expression of these genes in leaves,petioles and roots.The transcription levels of SoNRT1.3 and SoNRT1.5 in roots was significantly induced by nitrate supply,whereas,the transcription levels of SoNRT3 and SoNRT2 were significantly induced by nitrate deficiency in roots and their expression patterns were similar.2.The subcellular localization of the gene was carried out by Agrobacterium tumefaciens-infected tobacco transient expression method.It was found that SoNRT1.3and SoNRT2 were localized on the cell membrane.The five SoNRT genes in transgenic Arabidopsis thaliana was expressed under low and high nitrate treatment conditions though varied greatly between different tissues.In transgenic plants,the SoNRT1.3 and SoNRT1.5 in transgenic plants were mainly expressed in shoots,while the SoNRT1.9,SoNRT2 and SoNRT3 were expressed in both roots and shoots.Except for the SoNRT1.3-3 line,the shoot fresh weight of all the transgenic lines were about 2-fold higher than that of the wild type when under low nitrogen treatments.Overexpression of SoNRT1.5-7 also significantly increased the total nitrogen content of Arabidopsis thaliana shoots.High nitrate treatment significantly increased the shoot fresh weight of the transgenic SoNRT3-7 line.For transgenic Arabidopsis thaliana lines SoNRT1.3-3,SoNRT1.5-7,SoNRT1.9-1 and SoNRT3-7,their nitrate nitrogen contents decreased,while at the same time,their shoot fresh weights and total nitrogen contents increased or no changed.This indicated that these genes can be used as candidate genes for improving spinach quality with low nitrate contents.