Activation Of ERF6 To SRAGin Response To NaCl Stress In Arabidopsis

ACS gene family plays a very important role in response to salt stress in plants.It has been extensively reviewed that the ACS regulation on protein levels has been reported many literature.But how are the ACS genes regulated at transcription level need further research.This study takes wild-type plants,homozygous T-DNA insertion mutants srag,erf6 and ERF6-OE transgenic plants as experimental materials,exploring whether the Arabidopsis transcription factor ERF6 can regulate SRAG(Salt Relative ACS,SRA)expression in response to salt stress.Studies have shown that,there are significant differences between loss of function mutants erf6,srag and the wild type in seedling root length under salt stress.ERF6 and SRAG expressed in wild-type Arabidopsis whole plant,but the expression level at the root was significantly higher than other parts.Seedlings using 150 mM NaCl treatment,the whole plant in the wildtype Arabidopsis rapid and large increase in expression levels ERF6 and SRAG gene.The expression level of ERF6 and SRAG in wildtype increase rapidly in large amounts by treating the seedlings with 150 mM NaCl,which implies ERF6 and SRAG are involved in plant response to salt stress.Detecting by real-time PCR : the expression of SRAG decreased significantly in the loss of function mutant erf6,but the exression of ERF6 did not change in the loss of function mutant srag in return.We found that changes of ERF6 in gene expression level can result in SRAG changes simultaneously,and changes in gene expression of SRAG does not affect ERF6 expression,which implies that ERF6 may act in the upstream of SRAG,and can affect the expression SRAG.It has been studied that ERF6 can regulate the expression of the downstream resistance genes through a combination of DRE elements involving in the plant responses to salt stress in turn.It may means ERF6 may regulate the expression of SRAG acting as transcription factors.To verify this inference,we transform ERF6 and internal control vector into Arabidopsis leaves by using gene gun-mediated transient transformation under in vitro conditions.We found ERF6 can bind the promoter region of SRAG and start the downstream gene expression by detect dual luciferase activity.And then it was affirmed again by yeast one-hybrid experiments.We extracted and purifed ERF6 protein in vitro and synthesize a probe containing a sequence of DRE element in SRAG promoter.We showed that ERF6 could bind to the probe containing the DRE element by EMSA(electrophoretic mobility shift assay,EMSA),which proved again that ERF6 can be combined with SRAG promoter region.Eventually,using Co-Immunoprecipitation(Chromatin immunoprecipitation assay,CHIP)experiments,we proved that ERF6 can regulate the expression of SRAG in salt stress response by binding with DRE element in vivo conditions.In summary,the Arabidopsis transcription factor ERF6 can bind the DRE element of SRAG promoter region,and thus involved in the plant response to salt stress by regulating SRAG.