Analysis Of Stress Responsive Cis-elements In The Promoter Of CaWRKY27 And Identification Of Its Possible Interacting Protein

WRKY superfamily,which is one type of plat-specific transcription regulatory factors containing zinc finger structure,plays an important role in the process of adversity response in plants.It’s known that gene expression is regulated by the inter-coordination between cis-acting elements and trans-acting factors.Analysis of WRKY gene promoter and its potential interacting cis-acting factors is important for learning the disease-resistance molecular mechanism of induced promoter in higher plant.Full length of CaWRKY27 was isolated from a cDNA library of pepper previously,and can be induced by both biotic and abiotic stress such as high temperature,high-salinity,Ralstonia solanacearum infection and so on.In order to further clarify its expression regulation mechanism,we isolated CaWRKY27 upstream promoter from peppergenomic DNA by genome walking method and serial 5’deletions of CaWRKY27 promoter fused to the GUS reporter gene were constructed by Gateway technology.Through leaf-disc transformation method,the transgenic tobacco plants harboring the CaWRKY27promoter deletions were generated.The T1 transgenic tobacco plants and transient overexpression system were used for the activity measurement of GUS to analyzed the function of CaWRKY27 promoter.Through site-directed mutagenesis,we further confirmed the cis-acting element.The results are as follows:1.Using genome walking method,we isolated the upstream promoter region of CaWRKY27 from pepper genomic DNA(named pCaWRKY27,1608bp).The six 5’deletion of CaWRKY27 promoter fused to GUS reporter gene were constructed and named p1608,p1281,p1066,p888,p636,p528,p369.Then corresponding transgenic tobacco lines containing the six deletions were obtained through leaf-disc transformation method.2.The tobacco T1 transgenic lines were used for GUS activity measurement in response to biotic stresses,such as Rasltonia solanacearum inoculation,and abiotic stresses,such as high temperature and high salinity,as well as exogenously application of phytohormones,such as SA,JA,ET,and ABA.The data revealed that the GUS activity expression was enhanced in leaves treated with SA,Ralstonia solanacearum and high salinity in the-528～-369 bp region of CaWRKY27 promoter,in leaves treated with JA in the-888～-636 bp region,in leaves treated with ET in the-1066～-888 bp region,and in leaves treated with ABA in the--369～-1 bp region.However the GUS activity of the-1066～-888 bp region of CaWRKY27 promoter exhibited decrement after the treatment of high temperature.3.Transient overexpression assay indicated that GUS activity of-369～-1bp region of CaWRKY27 promoter can be induced by overexpression of CaWRKYl/58,and GUS activities of the-528～-369bp region and-888--636bp can be induced by overexpression of CaWRKY40 and CaWRKY27/6,respectively.4.Further site directed mutagenisis analysis of CaWRKY27 promoter indicated that the extract cis-element of CaWRKY27 promoter related to SA was TCA-element located between-415 bp and-406 bp;the MeJA-related cis-acting element is w-box which is located at-748～-742 bp;the ethylene-asscociated cis-acting elements are ERE and W-box which are located at-1051～1044 bp and-923～-918 bp;Cis-acting element ABRE which is located at-222～-218 bp was responded to ABA;Cis-acting element GT-1 boxs which are located at-686～-681bp and-438～-433 bp were responded to high salt;Cis-acting element W-box which is located at-455～-450 bp was responded to Ralstonia solanacearum.However,the high temperature responsive cis-acting element was not found.5.To confirm the exact W-box in the CaWRKY27 promoter that the WRKY protein bind to,site-directed mutagenesis and transient overexpression assays were performed.The result demonstrated that CaWRKYl can bind to 1W-box/2W-box/4W-box,CaWRKY40 binding to 2W-box,CaWRKY58 binding to 1W-box/4W-box,CaWRKY27 and CaWRKY6 binding with 1W-box,respectively,which trigger the expression of the downstream GUS reporter gene,however,the GUS reporter gene showed no induction when the corresponding w-boxes were mutated..6.Based on transient expression system and chromatin immunopricipitation assays,we found that the w-boxes of CaWRKY27 promoter not only can be bound by CaWRKY27 protein,but also other WRKY proteins and the binding abilities were lost or decreased when the w-boxes were mutated.Together,the result above indicated that the cis-element of the CaWRKY27 promoter can be bound by CaWRKY27 and other WRKY proteins in response to biotic and abiotic stresses,such as pathogen infection and salt stress,and plays an vital role in the defense reaction in response to stresses.