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. 2020 Apr 29;11(5):485. doi: 10.3390/genes11050485

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Identification of potential regulation of VviWRKY24 and VviWRKY32 on VviGT14. A. Yeast-one-hybrid assay showing that yeasts carrying a combination of effector and reporter vectors can grow on the medium without or with 400 ng/mL antibiotic aureobasidin A (AbA), whereas yeasts with AD and 3× w-box cannot grow on the media with 400 ng/mL AbA. The present result indicates that both VviWRK24 and VviWRKY32 can bind with the 3 repeat w-box sequence. B. Subcellular localization of VviWRK24 and VviWRKY32 in Arabidopsis protoplast. The graphs from left to right represent VviWRKY-eGFP, nucleus marker-mcherry, bright field, and the merge images. The present result indicates that both of the two TFs are specifically located in the cell nucleus. C. The reporter, effector and normalized control constructs used in the transcriptional activity assay. D. Transcriptional activation domain analysis of VviWRKY24 and VviWRKY32; The transcriptional activation functions of VviWRKY24 and VviWRKY32 were tested using the luciferase assay. The control represents empty pBD vector and its fold change is set as 1. VP16, an unusually potent transcriptional activator, is regarded as a positive control, and its activity change exceeds 200-fold. The error bars represent S.E.s from six biological replicates. Significant differences relative to the control are determined using the one-way ANOVA tests (**, P < 0.01). The present result indicates that both of the two TFs lack the transcriptional activation domain. E. The reporter, effector and normalized control constructs used in the dual luciferase assay. F. The transcriptional regulation assessment of VviWRKY24 and VviWRKY32 on the VviGT14 promoter activity in a transient expression system in Vitis vinifera L. “Chardonnay” cell suspension using dual luciferase assay. VviMYBA2 (BAD18978), a specific transcription factor in anthocyanin biosynthesis, is taken as negative control to test the reliability of this reaction system. The present result indicates that both of the two TFs have no transcriptional regulation effects on VviGT14 promoter. The data were shown as the mean ± SD from at least six biological replicates. ANOVA tests (*, P < 0.05. **, P < 0.01).