Cloning And Functional Analysis Of Vitis Vinifera WRKY26and WRKY27Promoters

European grape (Vitis vinifera L.) as one of the most important species of Vitis, is usedfor table grape and the wine, and its annual production accounts for about90%of the totalproduction of grapes in the world. Because of its poor stress resistant ability, Vitis vinifera L.is vulnerable to the injuries of environmental change and insect pests. The yield and qualityare difficult to be maintained. The researches showed that, in the process of plants’ stressresponse, the binding and regulations of specific transcription factors to stress resistance genepromoters regulated the expression of the downstream genes not only temporally but alsodifferentially, to increase the stress resistance of plants. WRKY proteins are a kind ofplant-specific transcription factors family. Most research showed that the WRKY proteinscould widely participate in various biotic and abiotic stress defense reactions. Therefore, weselected the two of WRKY genes (VvWRKY26and VvWRKY27) of Vitis vinifera L. to analysethe regulation ability of their promoters. The result will provide a theoretical foundation forthe stress resistance research of the grapes.Using the method of the Vitis vinifera L.’s whole genome sequences comparison, weselected about a1500bp sequence at the upstream of initiation codon as the target ofamplification. According to the information in the genome sequences we designed the primersand obtained the promoter sequences of VvWRKY26(1108bp) and VvWRKY27(1381bp)amplified by PCR.Two promoters were analyzed for plant gene promoters’ cis-element by using of theanalytic database PlantCARE. The results showed that VvWRKY26and VvWRKY27contain avariety of response elements which involved in plant development and biotic or abioticstresses, such as hormonal response element, salicylic acid response element, jasmonic acidresponse element, light sensitive response element, heat sensitive response element andendosperm development related response element, et al.According to the analysis results, we designed the upstream primers of VvWRKY26andVvWRKY27separately and cloned the promoter sequences with different lengths by the5 ’deletion method. These promoter sequences were named VvW26p-1, VvW26p-2, VvW26p-3,VvW26p-4and VvW27p-1, VvW27p-2, VvW27p-3respectively.By the enzyme digestions, we replaced the35’s promoter in the pBI-221expressionvector with the5’ deletion WRKY genes’ promoters to construct some transient expressionvectors regulated by the5’ deletion promoters. Then mediated by the PEG-Ca2+, Arabidopsisprotoplasts were transfected by the new expression vectors. The regulation ability of the twogroup of the5’ deletion promoters (VvWRKY26and VvWRKY27) were analysed by theexpression of LUC (Luciferase reporter gene) with the technology of transient fluorescenceexpression. The results showed that the reporter gene expression regulated by thedeferentially deleted promoters containing different activation and inhibition domains. Theresults might be the experimental references for the subsequent analysis of cis-element in theplant.