The Analysis Of Salt-responsive Genes And Functional Characterization Of Key KUP Transporters In Zygophyllum Xanthoxylum

Drought and irrigation-induced secondary soil salinity severely restrict the sustainable development of agriculture in northwest of China.The desert plants widely distributed in this area have developed unique mechanisms to adapt to harsh environments during the process of long-term evolution.To investigate the mechanism of these plants and explore tolerant gene resources are the foundation for the improvement of forages and crops production by genetic engeneering and the utilization of saline-alkalized land.Zygophyllum xanthoxylum is a salt-accumulating xerophyte,native to desert areas of China and Mongolia.Absorbing a great quantity of Na~+and maintaining the stable of K~+concentration in low-Na~+and K~+soil are the effective strategies for Z.xanthoxylum to adapt harsh environments.Thus,Z.xanthoxylum is more appropriate model for studying salt and drought tolerance compared with glycophytes.At present,the molecular mechanism of salt tolerance in Z.xanthoxylum has not been studied systematically,meanwhile,the function of KUP/HAK/KT transporter in Z.xanthoxylum remains unclear.Moreover,the synergistic effect of key Na~+and K~+transporters at the whole plant level of Z.xanthoxylum remains to be further explored.The aims of this Ph.D research,therefore,are to reveal unique genetic adaptations in Z.xanthoxylum using comparative transcriptome analysis,further characterize the function of key KUP/HAK/KT transporter based on transcriptome,as well as investigate the expression of key Na~+and K~+transporters.The main findings are as follows:1.Transcriptome profiling by RNA-Seq was used for comparing gene expression in roots of Z.xanthoxylum and A.thaliana under 50 mM NaCl treatments.GO functional annotation and KEGG metabolic pathway analysis suggested that 50 mM NaCl was perceived as a stimulus for Z.xanthoxylum whereas a stress for A.thaliana.Exposure to 50 mM NaCl caused metabolic shifts toward gluconeogenesis to stimulate growth of Z.xanthoxylum,but triggered defensive systems in A.thaliana.Compared with A.thaliana,a vast array of ion transporter genes was induced in Z.xanthoxylum,revealing an active strategy to uptake Na~+and nutrients from the environment.An ascorbate-glutathione scavenging system for reactive oxygen species was also crucial in Z.xanthoxylum,based on high expression of key enzyme genes.Finally,key regulatory genes for the biosynthesis pathways of abscisic acid and gibberellin showed distinct expression patterns between the two species and auxin response genes were more active in Z.xanthoxylum compared to A.thaliana.Our results provide an important framework for understanding unique patterns of gene expression conferring salt tolerance resistance in Z.xanthoxylum.2.The full-length cDNA of ZxKUP7,a potassium transporter encoding gene,was cloned.ZxKUP7 belonging to Clade?of KUP family was closely related to AtKUP7with 76.4%similarity in amino acid sequence and localized in the plasma membrane.ZxKUP7 was mainly expressed in the root and was significantly induced by KCl treatment,NaCl treatment and osmotic stress.We constructed the complementation lines of atkup7 using ZxKUP7.Salt tolerance and drought resistance of transgenic lines were analyzed.In water culture experiments,atkup7 was sensitive to salt,with K~+concentration in roots and shoots decreased and Na~+concentration increased.Therefore,the homeostasis of K~+and Na~+was destroyed.The results of salt stress conducted under soil culture showed the K~+concentration in rosette leaves of atkup7 was reduced,while Na~+concentration was increased,resulting in the poor growth of atkup7.However,K~+concentration in complementation lines was increased,furthermore,photosynthetic was improved.Under drought treatment,K~+accumulation and tissue water content in rosette leaves of atkup7 were decreased.However,that in the complementation lines were higher than atkup7.These results indicated that the founction of ZxKUP7 is similar to AtKUP7,and ZxKUP7 may involve in K~+uptake and plays important role in maintaining K~+and Na~+homeostasis.3.The full-length cDNA of the potassium transporter gene ZxKUP6 was cloned.ZxKUP6 belonging to Clade?of KUP family was closely related to AtKUP6 with 75.6%similarity in amino acid sequence and localized in the plasma membrane.ZxKUP6mainly expressed in the root was significantly induced by KCl treatment,NaCl treatment and osmotic stress.Overexpression of ZxKUP6 improved the primary root length of transgenic Arabidopsis significantly under osmotic stress,and increased the K~+concentration by 30%in rosette leaves of transgenic lines under drought treatment.Meanwhile,the transgenic lines showed better growth,higher relative water content and chlorophyll content.Based on the above results,ZxKUP6 might play a role in K~+translocation from root to shoot,furthermore increase the accumulation of K~+in shoot.4.The expression patterns of key Na~+and K~+transport genes under 50 mM NaCl treatment were studied by ZxNHX1-RNAi lines and wild-type.When ZxNHX1 was silenced,under 50 mM NaCl treatment,the expression level of ZxHKT1 continued to increase in roots of ZxNHX1-RNAi line,while the transcription of ZxSOS1 down-regulated.As a result,the capability of ZxHKT1 to unload Na~+from xylem sap to xylem parenchyma cells was increased,while the capability of ZxSOS1 to load Na~+into xylem was decreased.Eventually,Na~+was inhibited to transport from root to shoot in ZxNHX1-RNAi lines.ZxHKT1;2 mediated Na~+absorption and ZxHKT1;3 involved in Na~+loading into mesophyll cells were also significantly induced in leaves of wild-type,but was down-regulated in ZxNHX1-RNAi line.Moreover,ZxNHX1 indirectly regulates the K~+absorption and transport.Under 50 mM NaCl treatment,ZxAKT1,ZxKUP7,ZxKUP6 and ZxSKOR were inhabited in ZxNHX1-RNAi line.Therefore,we suggested that the abilities of to absorb K~+and transport K~+from root to shoot in ZxNHX1-RNAi line were weakened.In summary,salt-accumulating xerophyte Z.xanthoxylum has a unique salt tolerance mechanism.ZxKUP7 may involve in K~+uptake and ZxKUP6 might play a role in K~+translocation from root to shoot.Both of the two transporer play important roles in K~+and Na~+homeostasis.Key Na~+and K~+transporters synergistically modulate Na~+and K~+uptake and tranlocation at the whole plant level of Z.xanthoxylum.