Analysis Of Salt Tolerance Function Of ThABF2 From Tamarix Hispida

ABF/AREB(ABRE-binding facto/ABA-response element binding factor)proteins are a class of transcription factors widely found in plants,belongs to the A subfamily of bZIP-type transcription factors,and mainly play a crucial role in the growth and development and other aspects.In this study,a ABF gene that was involved in the resistance to salt stress response was identified from the transcriptome of Tamarix hispida.The gene named ThABF2 and the open reading frame(ORF)is 1110 bp in length,encoded 370 amino acids.The relative molecular mass is 82.3 kDa,and the theoretical isoelectric point(PI)is 5.02.The pBI121-GFP-ThABF2 vector was constructed,the subcellular localization results showed that ThABF2 protein was expressed in the nucleus.Then the pGBKT7-ThABF2 vector was constructed,and Y2H yeast competent cells were transformed and screened by SD/-Trp/-His/-Ade/X-?-Galmedium.The results showed that the ThABF2 transcription factor has a transcriptional activation activity.To study the salt tolerance of the ThABF2 gene,the recombinant vectors pROKII-ThABF2 and pFGC5941-ThABF2 were constructed to carry out the transient transformation of T hispida,and the transient infection of T.hispida by pROKII empty vector was used as the control.Futhermore,we analyzed and compared the physiological staining and physiological indexes after NaCl stress.The results showed that the over-expressing strains have the lightest staining after stress,and the deepest staining of the inhibiting-expressing lines,the H2O2 content,MDA content and relative conductivity in the overexpressed plants(OE)were the lowest,and they in the RNAi plants were relatively highest.POD activity,SOD activity and chlorophyll content were the highest in the overexpressed plants(OE),and they were the lowest in the RNAi plants.These indicated that ThABF2 in transgenic T.hispida plants increased the POD and SOD activity,decreased intracellular H2O2 and O2-content,and then improved the plants' salt tolerance to stress.To further examine the results of transient infection,the pROK?-ThABF2 gene was transfered into wild-type(WT)Arabidopsis.And two T3 homozygous lines were selected to further analyze the salt stress function.After NaCl stress,the growth indexes which were analyzed and compared between the transgenic and wild-type Arabidopsis.The results showed that the germination rate,root length and fresh weight of the overexpressed lines after salt stress were significantly higher than those of WT,and the growth was better than that of the control.Further,the the physiological staining and physiological indexes of the transgenic and wild-type control lines before and after salt stress were also analyzied.The results were consistent with the transient transformation of T.hispida.The ThABF2 transgenic Arabidopsis plants were stained lightest,and the H2O2 content,MDA content and relative conductivity in the overexpressed plants(OE)were the lowest,POD activity,SOD activity and chlorophyll content were the highest.It was shown ThABF2 gene can improve the salt tolerance of transgenic Arabidopsis.The TF-Centered Y1H technology(transcription factor-centric yeast one-hybrid technology)was used to identify the cis-acting elements that ThABF2 transcription factors may recognize,and verified by yeast one-hybrid method.The results showed that the ThABF2 gene can specifically recognize the "CGTAAT" sequence,laying a foundation for further study of its downstream target genes and regulatory mechanisms.