Analysis Of Salt Tolerance Function Of ThSZF3 From Tamarix Hispida

Zinc Finger Proteins(ZFP)are important transcriptional regulators of plants and participate in many important biological processes such as morphogenesis,pollen development,stress response,and embryo development.In previous studies,it was found that the ThZFP gene may be the salt-tolerant gene in Tamarix hispida.Therefore,the ThZFP gene was selected for further study of salt stress resistance and preliminary analysis of its salt tolerance regulation mechanism.In this study,the 1176 bp sequence of ThSZF3 gene was selected from the transcription database of.hispida.The open reading frame of ThSZF3 cDNA was 1176 bp which encoding 391 amino acids.The relative molecular weight of the ThSZF3 protein was 97.26kDa,with the isoelectric point of 5.00.The subcellular localization results showed that ThSZF3 protein was expressed in the nucleus.The over-expression(OE),RNAi and control group(CON)of trangenic plants were obtained by transient transformation into.hispida.Futhermore,we analyzed and compared the physiological indexes such as DAB,NBT and Evas Blue staining and POD,SOD,PRO,MDA,H2O2 content and relative conductivity in these transgenic plants after NaCl treatment.The results showed that over-expression of ThSZF3 in T.hispida plants had the highest POD,SOD,PRO,but the lowest O2-,H2O2 and MDA accumulation,minimal cell death,and the lowest electrolyte leakage rate among the three kinds of transgenic plants.Conversely,the RNAi-silencing transient transformation plants were display the opposite physiological dynamic trendaney.The results showed that ThSZF3 could increase the activity of antioxidant enzymes in.hispida,promote the elimination of reactive oxygen species,and reduce the cell membrane damage,thus increase ability of the.hispida to high salt tolerance.To further verify the results of transient expression of ThSZF3 gene in.hispida,ThSZF3 overexpression vector was transformation into wild-type(WT)Arabidopsis thaliana under salt stress.And two T3 homozygous lines were selected for further analysis the function.After stress with 150 mM NaCl,the growth indexes such as seed germination rate,root length,fresh weight and growth vigor were analyzed and compared between the transgenic and WT Arabidopsis thaliana.The results examined that the germination rate,root length and fresh weight of the overexpressed lines were significantly higher than those of WT after salt stress.And the growth vigor was better than the wild-type control line.These results were indicated that the salt tolerance of transgenic lines were significantly increased.Furthermore,the physiological indicators such as DAB,NBT,Evans blue staining and POD,SOD,PRO,MDA,H2O2 content,relative conductivity and other indicators of the transgenic and wild-type control lines before and the after salt stress were also analyzed.The ThSZF3 transgenic Arabidopsis thaliana plants were stained lighter,the POD,SOD,PRO were dramatically higher than the control,and the MDA,H2O2 content and relative conductivity values were significantly lower than the control.It demanstrated that ThSZF3 gene could increase the salt tolerance in the transgenic plants by reducing the active oxygen content,and protecting cell membrane integrity.Additionally,using the TF-yeast one-hybrid,novel interaction patterns were detected.We identified four motifs that were specifically bound by ThSZF3,including a novel motif SBS and three known motifs(Dof,CAAT-box,G-box).The motif sequence "CTGCC" was found by yeast one-hybrid.Transcriptome sequencing was used to identify the different expression pattern of ThSZF3,over-expression,suppress-expression and control of ThSZF3 in.hispida.Compare to the control,there are 22 genes showed up-regulated in the over-expression of ThSZF3,and 140 genes down-regulated between ThSZF3 suppress-expression line and control.There are 6 genes shared between the two groups.It was found that the "CTGCC" motif was present in the four gene promoter sequences,and the promoter sequence of ThEIN2(Ethylene-insensitive protein 2)was selected.The results of yeast one-hybrid showed that the "CTGCC"motif could regulate the binding of ThEIN2 gene to ThSZF3,which might be a downstream gene of ThSZF3.