Identification And Functional Verification Of NPF And NRT2 Gene Families In Quinoa (Chenopodium Quinoa Willd.)

Chenopodium quinoa Willd.,an annual dicot plant in the genus Amaranthaceae,has excellent stress tolerance(salinity,drought,low temperature,and barren tolerance)and can be grown on marginal soils.Nitrogen(N)is an essential mineral nutrient for plants and is considered a key factor limiting crop yield and quality.NPF and NRT2 gene family regulate nitrogen uptake by terrestrial plants.NRT2 is a high affinity nitrate transporter protein that mediates the absorption of low concentrations of nitrate;NPF is a low affinity nitrate transporter protein that plays a role when the external nitrate concentration is greater than 1mmol/L.At NPF2.13 is located in the phloem to transport nitrate from old leaves to tissues that require nitrogen,which is crucial for the reuse of nitrate.In this study,we first identified the members of NPF and NRT2 gene family in the whole genome of quinoa,and carried out bioinformatics analysis such as phylogenetic analysis,gene structure,chromosome location and promoter function prediction,as well as expression pattern analysis under different tissues and abiotic stress;Secondly,the physiological and molecular response characteristics of different genotypes of quinoa to low nitrogen stress were compared;Finally,three genes with high homology with Arabidopsis At NPF2.13 were further screened for in-depth research.The main results are as follows:(1)The quinoa genome contains 104 NPF and 16 NRT2 genes.CqNPF is divided into 8 subfamilies based on similarity of gene structures and conservation of protein motifs;The collinearity analysis showed that the expansion of the members of the two families was mainly driven by fragment replication and was affected by purification selection;Promoter element analysis showed that the two family members containe abiotic stress,growth and development and hormone response elements.(2)Different tissue expression patterns showed that the NPF7 subgroup of quinoa was mainly expressed in the apical meristem and inflorescence,the NPF5 subgroup was mainly expressed in the seedling stage,the NPF1 and NPF2 subgroups were mainly highly expressed in flowers and immature seeds,petioles and leaves,and the NPF6,NPF8 and NPF4 subgroups were expressed in all tissues;There are 2 constitutive expression genes and 3 tissue specific expression genes in CqNRT2.The expression pattern of abiotic stress showed that both CqNPF and CqNRT2 gene family were responsive to drought,salt,high temperature and low phosphate stress,but there were differences between aboveground and underground tissues.(3)Comparing the physiological and molecular responses of different genotypes of quinoa to low nitrogen stress,the results showed that both varieties responded to the oxidative damage caused by low nitrogen stress by increasing the activities of antioxidant enzymes and nitrogen assimilation enzymes,and the content of osmotic regulators proline and chlorophyll.The expression levels of CqNPF2 and CqNRT2 genes in leaves and roots were significantly increased.Different genotypes have different abilities to maintain growth and development,and the coastal lowland variety Faro has stronger tolerance to low nitrogen stress.(4)The results of heterologous overexpression of CqNPF2.13,CqNPF2.19,and CqNPF2.21 genes in Arabidopsis thaliana,as well as the substitution of mutants,showed that the overexpression lines were more adaptable to low nitrogen stress,with leaf area and plant height superior to the wild-type.The bolting time was earlier,and the growth and development phenotype of the mutant substitution lines could be restored to the level of the wild-type.The chlorophyll content and activities of nitrogen assimilation enzymes(GS and NR)in the overexpression strain were significantly higher than those in the wild type.The above experimental results indicate that there are differences in the physiological and molecular responses of different genotypes of quinoa to low nitrogen stress.The phenotypic and physiological indicators of heterologous overexpression in Arabidopsis showed that CqNPF2.13,CqNPF2.19,and CqNPF2.21 can enhance the tolerance of quinoa to low nitrogen.The research results can be used to select quinoa varieties with high nitrogen utilization efficiency and improve crop tolerance to low nitrogen,reduce nitrogen fertilizer use,and improve the utilization efficiency of saline alkali land,which has important potential application value.