Functional Analysis Of Tobacco High-Affinity Nitrate Transporter Gene NtNRT2.5

Nitrogen is in great demand in plants and participates in the synthesis of many macromolecular substances such as nucleic acids,hormones,and proteins in plants.Plants must obtain a sufficient supply of nitrogen from the environment to ensure their own growth and development,but various biological and non-biological factors limit the absorption of nitrogen by plants.Therefore,plants have formed a series of mechanisms to adapt to low-nitrogen stress,by inducing or inhibiting the expression of nitrogen-related genes and proteins,and then regulating the absorption,redistribution,and assimilation of nitrogen to maximize the optimal utilization of nitrogen nutrition.The nitrate transporter is directly related to nitrogen absorption and utilization.The functional study of the tobacco nitrate transporter gene NtNRT2.5 can provide a new theoretical basis for the selection of tobacco varieties with high nitrogen utilization.In this study,bioinformatics prediction analysis was performed on the nitrate transporter gene NtNRT2.5,and qRT-PCR was used to analyze the nitrogen regulation characteristics of tobacco NtNRT2.5.We further used NtNRT2.5 gene promoter fused with GUS reporter gene tobacco for GUS tissue staining.We created NtNRT2.5 over-expression transgenic tobacco materials,and through nutrient solution hydroponics,we analyzed physiological and biochemical indicators of NtNRT2.5 over-expression tobacco under different concentrations of nitrogen treatments,such as the biomass,primary root length,nitrogen accumulation and nitrogen utilization efficiency,soluble protein and nitrate concentration,key enzyme activity of nitrogen metabolism,relative transcript levels of nitrogen metabolism-related genes,photosynthetic characteristics,photosynthetic pigment concentration,antioxidant capacity,and relative transcript levels of antioxidant-related genes,etc..The main results are as follows:(1)The results of bioinformatics analysis showed that the open reading frame of the NtNRT2.5 gene was 1509 bp in length,encoding 502 amino acid residues,and the theoretical isoelectric point was 8.50.The secondary structure of protein encoded by NtNRT2.5 gene is mainly alpha helix,and the protein contains 11 transmembrane structural regions.There was a central hydrophilic region between the 6th and 7th transmembrane regions to divide the transmembrane domain into two parts.The phylogenetic tree results showed that tobacco NtNRT2.5 had a closer evolutionary relationship with Arabidopsis AtNRT2.5,and the subcellular localization results showed that the protein encoded by NtNRT2.5 localized in the plasma membrane.qRT-PCR results showed that the NtNRT2.5 gene was mainly expressed in roots,stems and leaves of tobacco,especially in senescent leaves,and the expression was enhanced by low nitrogen induction.The results of GUS tissue staining showed that under both normal nitrogen supply and low nitrogen conditions,the GUS reporter gene was detected in the roots,stems and senescent leaves of NtNRT2.5 gene promoter fused with GUS reporter gene tobacco,which showed a blue color,and the expression was more pronounced under low nitrogen conditions.The results indicate that NtNRT2.5 is a high-affinity nitrate transporter gene,and the expression of this gene is enhanced by low nitrogen induction,and may be involved in the absorption,transport and reuse of nitrate.(2)We used NtNRT2.5 over-expression transgenic tobacco as materials to study the response of NtNRT2.5 to different concentrations of nitrogen.It was found that under the conditions of low nitrogen stress,wild-type tobacco grew dwarf,had poor root development,elongated primary root,and low biomass.The nitrogen accumulation and nitrogen use efficiency of tobacco leaves were decreased.It showed that low nitrogen stress inhibited the absorption and utilization of nitrogen in tobacco,which was not conducive to the growth and development of tobacco.However,the biomass and primary root length of NtNRT2.5 over-expression strains,as well as the nitrogen accumulation and nitrogen utilization efficiency of tobacco leaves were significantly higher than those of wild-type tobacco.(3)In order to explore the reasons for the changes in nitrogen utilization efficiency of tobacco leaves,we further measured the contcentration of soluble protein and nitrate,the activities of NR(nitrate reductase)and GS(glutamine synthetase),and the expression levels of genes related to nitrogen metabolism of tobacco leaves.The results showed that under both normal nitrogen supply and low nitrogen conditions,compared with wild-type tobacco,the soluble protein contcentration and NR activity of the NtNRT2.5 over-expression tobacco leaves were significantly increased.Under low nitrogen treatments,the nitrate contcentration of NtNRT2.5 over-expression tobacco leaves was significantly decreased,and the GS activity was significantly increased.NR and GS are key enzymes of plant nitrogen metabolism,and soluble protein is the product of nitrogen assimilation.NR reduces the nitrate nitrogen absorbed by plants into ammonium nitrogen for plant growth.GS is the main assimilation enzyme for plants to produce ammonium salts.The ammonium salts and nitrogen transport compounds produced by photorespiration and protein decomposition can be assimilated by GS again,which can improve crop nitrogen utilization efficiency.The relative transcript levels of nitrogen metabolism-related genes NtNIAl,NtNiR,NtGS3 and NtAMT2 in tobacco leaves were analyzed by qRT-PCR.The results showed that the over-expression of NtNRT2.5 enhanced the transcript levels of NtNIAl,NtGS3 and NtAMT2 in the nitrogen absorption and assimilation pathways of tobacco leaves,and made tobacco leaves have a more reasonable nitrogen distribution mechanism and a more efficient nitrogen utilization capacity in low nitrogen conditions.(4)Nitrogen is a component of chlorophyll,and plant photosynthesis requires the participation of chlorophyll.The abundance or deficiency of nitrogen in the environment affects the synthesis of chlorophyll and thus affects photosynthesis.Therefore,we studied the effect of nitrogen stress on the photosynthetic capacity of tobacco leaves.Compared with wild-type tobacco,the chlorophyll concentration of the NtNRT2.5 over-expression tobacco leaves were significantly increased,the photosynthetic index Pn(net photosynthetic rate),Tr(transpiration rate)and Gs(stomatal conductance)were significantly increased,and Ci(intercellular Carbon dioxide concentration)decreased significantly,indicating that the over-expression of NtNRT2.5 enhanced the photosynthetic capacity of tobacco leaves.(5)When plants are under adversity stress conditions,the dynamic balance of reactive oxygen species in cells is broken,triggering disorders of metabolic pathways operating in organelles such as chloroplasts and mitochondria,leading to oxidation of proteins,chlorophyll,lipids,nucleic acids,etc.,which affect enzyme activity.Therefore,we studied the antioxidant capacity of NtNRT2.5 over-expression tobacco under nitrogen stress.Compared with wild-type tobacco,NtNRT2.5 over-expression tobacco leaves had lighter NBT staining and lower MDA concentration.Furthermore,the relative transcript levels of antioxidant-related genes NtSOD and NtGPX were significantly increased,and relative transcript levels of reactive oxygen generation-related genes NtRBOH was greatly reduced in a nitrogen-deficient environment.Over-expression of NtNRT2.5 enhanced the antioxidant capacity of tobacco leaves.To sum up,the tobacco NtNRT2.5 is a high-affinity nitrate transporter protein gene,and its expression is enhanced by low nitrogen induction.It participates in the absorption,transport and reuse of nitrate,improves the nitrogen utilization efficiency of tobacco leaves,enhances the photosynthetic capacity and antioxidant capacity of tobacco leaves,and promotes the growth of tobacco.