Cloning Of NAXT Homologous Genes And Functional Analysis Of One Member PcNPF2.7 In The Xerophyte Pugionium Cornutum

Soil salinization severely restricts the sustainable development of agricultural and livestock production in North China,and poses a serious threat to the ecological environment.The xerophytes that distributed in desert region,have evolved a series of unique mechanisms of stress tolerance during the long-term evolution process,and contain rich stress-resistant gene resources.To further explore the stress tolerance mechanisms and relevant molecular basis of xerophytes,and excavate important genes closely related to stress tolerance,will provide a theoretical basis for the improvement of crops and forages.Pugionium cornutum,which is widely distributed in the desert and semi-desert areas of Northwest China,is a typical succulent xerophyte with excellent drought and salt tolerance.Previous studies have shown that P.cornutum could accumulate a large amount of Cl^-in shoot for osmotic adjustment under salt stress,and meanwhile remain stable NO₃^-concentration in shoot.Therefore,maintaining the relative stability of NO₃^-/Cl^-homeostasis in shoot is one of the strategies for P.cornutum to adapt to saline environments,but the molecular mechanisms underlying the stable NO₃^-/Cl^-homeostasis in shoot of P.cornutum under salt stress remains unclear.Studies have shown that the nitrate efflux transporters NAXTs plays important roles in regulating NO₃^-/Cl^-homeostasis,however,the related studies only focused on the model plant Arabidopsis thaliana,in which the NO₃^-concentration decreased significantly with the increase of Cl^-content in plants under salt stress.It is unclear how NAXT family members from the xerophyte P.cornutum function in maintaining shoot NO₃^-/Cl^-homeostasis and regulating the salt tolerance.Therefore,in the present study,NAXT family homologous genes were cloned from the desert xerophyte P.cornutum,and their expression pattern under salt treatments were analyzed;the tissue and subcellular localization of PcNPF2.7,one member of NAXTs family in P.cornutum,were confirmed,and its function in the accumulation of ions including NO₃^-under salt treatments and the salt tolerance were analyzed by overexpressing it in A.thaliana.The main results are as follows:1.The NAXT family member genes,named as PcNPF2.6 and PcNPF2.7,were cloned from P.cornutum.The full length of the two c DNAs is 1863 and 1815 bp,which encodes 562 and 561 amino acids,respectively.Both of these two genes were mainly expressed in roots,and their expression level was significantly induced by Na Cl,Na NO₃ treatments and osmotic stress.2.PcNPF2.7 was mainly expressed in the root stele,and the encoding protein was located at the plasma membrane.After the stelar-specific overexpression of PcNPF2.7in Arabidopsis,the shoot NO₃^-concentration and NO₃^-/Cl^-ratio was significantly increased under salt stress,resulting in an enhancement of salt tolerance.The expression abundance of At NPF2.3 and At NPF7.3,the two key functional genes involved in the transport of NO₃^-from root to shoot,in the transgenic lines overexpressing PcNPF2.7showed the same level as that in wild-type plants,indicating that PcNPF2.7 might mediate the long-distance transport of NO₃^-from root to shoot,and thus function in maintaining shoot NO₃^-/Cl^-homeostasis.3.After the stelar-specific overexpression of PcNPF2.7 in Arabidopsis,the expression level of At HKT1;1,which mediates the unloading of Na⁺from xylem,was significantly induced in roots,and thus the shoot Na⁺concentration was significantly decreased.This might be another important reason why overexpression of PcNPF2.7significantly enhanced the salt tolerance in Arabidopsis.These results indicate that PcNPF2.7 might be involved in the transport of NO₃^-from root to shoot and thus affects the shoot NO₃^-/Cl^-homeostasis,and meanwhile decrease shoot Na⁺concentration,therefore plays a vital role in salt tolerance of plants.