Functional Analyses Of Salt Stress Related Gene TaCIPK25 In Wheat

Abiotic stresses such as high extreme temperatures,salinity and drought,seriously affect plant growth and crop yield.Wheat is one of the most important food crops in the world,identification and functional analysis of wheat stress reposive genes has important theoretical and practical significance on revealing the molecular mechanism underlying plant response to stresses and wheat stresses tolerance breeding.Ca2+,as a second messenger,plays important roles in signal transduction in plant stress response.Calcineurin-B like protein(CBL),one of the plants unique Ca2+ sensors,when combined with Ca2+,can activate its interacting protein kinase(CIPK),and then initiate a series of stress response through the phosphorylation of downstream target molecules.Previously,systematical analysis of CIPK gene family identified 79 CIPK genes in wheat genome in our laboratory,but the biological functions of most of the members remains unclear.In the present work,based on expression pattern analysis result of TaCIPK25,we carried our functional analysis of TaCIPK25 in transgenic Arabidopsis and wheat in response to salt stress by gene over-expression technology.The main research contents and results are as follows:1)TaCIPK25 gene expression profilingUsing real-time quantitative fluorescence RT-PCR,we analyzed the expression patterns of TaCIPK25 in different organs of wheat at different developmental stages.The results showed that TaCIPK25 was expressed in wheat root,stem,leaf,coleoptile,pistils and stamens.The highest expression level was in coleoptile,followed by the stem,the lowest expression was in stamens;Wheat seedlings were treated with ABA and NaCl,the expression of TaCIPK25 in leaves and roots were affected,and showed a similar trend,that is,in leaves the expression of TaCIPK25 showed a first downward and then upward trend,while in root TaCIPK25 was down-regulated,indicating that TaCIPK25 may participate in the ABA and NaCl salt stress signaling pathway.2)Subcellular localization and co-localization of TaCIPK25 and TaCBLlIn our previous study,we used the yeast two hybrid assay to analyze the interaction between TaCIPK25 and 8 TaCBLs,and found that TaCIPK25 and TaCBLl interacted with each other.In order to further study their interaction in plant,we constructed pBI121-TaCIPK25-GFP,pBI121-TaCBL1-GFP plasmids for subcellular localization and YCE-TaCIPK25,YNE-TaCBL1 plasmids for bimolecular fluorescence complementation.By using the method of particle bombardment,the above constructs were transiently expressed in onion epidermal cells,and results showed that TaCIPK25-GFP was distributed throught the whole onion epidermal cells,while TaCBL1 was specificly located on the plasma membrane.The results of bimolecular fluorescence comple-mentation experiment showed that TaCBL1 and TaCIPK25 were co-localized on the plasma membrane,indicating that TaCIPK25 and TaCBLs could interact each other in plant cell,and TaCIPK25 was recruited to the plasma membrane.3)The functional analysis of TaCIPK25 gene in transgenic Arabidopsis.In order to analyze the function of TaCIPK25 gene in response to stress,TaCIPK25 was transformed into Arabidopsis by Agrobacterium mediated method,and the positive transformation lines were obtained,and the phenotype of T3 transgenic Arabidopsis were analyzed under salt stress.The results showed that overexpression of TaCIPK25 could increase the sensitivity of Arabidopsis to salt stress.The content of Na+ in the transgenic plants was significantly higher than that in the wild type plants.It was inferred that the sensitivity of TaCIPK25 over-expression transgenic Arabidopsis might be resulted from excessive accumulation of Na+ in cells.4)The function analysis of TaCIPK25 in transgenic wheatIn order to study the function of TaCIPK25 in wheat under salt stress,TaCIPK25 over-expression transgenic wheat was obtained by particle bombardment of wheat immature embryos,and the phenotype of transgenic and wild wheat under salt stress were analyzed.The results showed that TaCIPK25 over-expression could also increase the salt sensitivity of transgenic wheat.Na+ content in transgenic wheat was also significantly higher than that in wild type.Through the determination of Na+/H+ flow in wheat root cells,it was found that Na+ efflux in transgenic lines decreased significantly,therefore,we speculated that the TaCIPK25 might weaken the activity of Na+/H+ antiporter on cytoplasmic membrane,resulting in the blocked Na+ efflux.5)Analysis of the regulatory mechanisms of TaCIPK25 promoterIn order to study the regulation of TaCIPK25 expression,we cloned the promoter of TaCIPK25 and analyzed its conserved cis-elements.It was revealed that the TaCIPK25 promoter contained 5 WRKY binding sites(W-box element).In order to study the regulation of WRKY transcription factor on the expression of TaCIPK25 gene,we selected three TaWRKYs(TaWRKYl,TaWRKY9 and TaWRKY44),which were representative of the three WRKY sub-families.By Yeast one hybrid assay,DPI-ELISA experiment,tobacco transient expression experiments and gene expression analysis,we found that TaWRKY9 could bind to the W5-box on the promoter of TaCIPK25 gene and negatively regulates the expression of TaCIPK25 through an ABA dependent pathway under salt stress conditions.