Functional Study On Salt Tolerance And Drought Resistance Of NAC7 Gene From Tamarix Hispida

Plant specific NAC(NAM,ATAF1/2 and CUC2)transcription factors(TFs)play important roles in plant xylem development,seed development,hormone synthesis and abiotic stress response.In previous study,ThNAC7 gene was cloned from Tamarix hispida,which was proved that had salt tolerance in transgenic Arabidopsis thaliana.Here,the salt tolerance and drought resistance of ThNAC7 gene were further studied,which laid a foundation to clarify the stress tolerance mechanism of ThNAC7.In this study,multiple sequence alignments showed that ThNAC7 conserved domain in N-terminus.Phylogenetic tree analysis was discovered that ThNAC7 was closely related to ANAC091,ANAC062 and ThNAC35.The fluorescent localization expression vector pBI 121-GFP-ThNAC7 were constructed,and subcellular localization showed that ThNAC7 was a nuclear localized protein.ThNAC7 gene overexpression vector(pROK ?-ThNACT)and inhibitory expression vector(pFGC5941-ThNAC7)were constructed,both them and pROK? vector were used by Agrobacterium-mediated efficient transient transformation system to obtain overexpressing ThNAC7(OE)and inhibitory expression ThNAC7(IE)transiently transformed T.hispida seedlings,which were generated to investigate abiotic stress tolerance via the gain-and loss-of function.Under stress conditions,the results showed that,the superoxide dismutase(SOD)and peroxidase(POD)enzyme activities and proline(PRO)content of OE were highest in three transgenic plants,the hydrogen peroxide(H2O2)content,malondialdehyde(MDA)content,electrolyte leakage and water loss rate were the lowest.In contrast,the activities of POD,SOD enzyme and PRO content in IE plant were the lowest,H2O2 content,MDA content,electrolyte leakage and water loss rate were the highest.In order to further verify the results of transient transformation of T.hispida,the pROK ?-ThNAC7 overexpression vector was stably transformed into Arabidopsis thaliana using the floral dip method,and T3 generation homozygous strains were selected for futher study.Compared with wild type,overexpressing ThNAC7 stably transformed A.thaliana also showed higher activities of POD,SOD enzyme and PRO content,lower H2O2 content,MDA content,electrolyte leakage and water loss rate.These results showed that ThNAC7 protected the integrity of cell membranes by reducing the accumulation of ROS,improving the salt and drought tolerance in transient transformed T.hispida and stably transformed A.thaliana plants.Under salt stress and normal conditions,ThNAC7 gene overexpression(OE5)and wild type A.thaliana were analyzed by RNA-seq.The results showed that 272 differentially expressed genes were obtained,185 up-regulated and 87 down-regulated under normal conditions.Under salt stress conditions,a total of 673 differentially expressed genes were obtained,with 453 up-regulated and 220 down-regulated.Twelve genes(6 up-regulated and 6 down-regulated)were selected randomly for research,using qRT-PCR and R2 correlation analysis to verify the results of transcriptome data.Among the obtained differentially expressed genes,there were a variety of coding proteins and transcription factor genes which related with stress response,coding proteins genes such as peroxidase,lipid transfer protein and MAP kinase.Transcription factor genes such as ERF,MYB,HSF,DREB,TCP,NAC,bZIP,WRKY,etc.,indicated that ThNAC7 gene can regulate the expression of genes which related with abiotic stress.