Cloning and functional analysis of the VdWRKY26 gene in Vitis davidii

【Objective】Vitis davidii. is an important wild grape germplasm resource in southern China. The organic acids in its fruits primarily accumulate in the form of tartaric acid, malic acid and citric acid in the vacuoles during the early stages of fruit development, a process primarily influenced by aluminum- activated malate transporter (ALMT9), vacuolar membrane dicarboxylate transporter (tDT), and vacuolar membrane proton pump. This study aimed to investigate the function of the VdWRKY26 gene in the growth and development of Vitis davidii fruits. The coding region (CDS) and promoter sequence of the VdWRKY26 gene were cloned from Xiangci No. 1, and its function and promoter activity were further analyzed.【Methods】The Pearson correlation coefficients between VdWRKY26 gene expression data and organic acid content were calculated using the R language packages GGally and ggplot2. Using cDNA from Vitis davidii fruits tissue as a template, primers were designed via the Oligo7 website for PCR amplification. The CDS region of the VdWRKY26 gene was cloned via reverse transcription polymerase chain reaction (RT- PCR). The phylogenetic tree and multiple amino acid sequence alignments of the VdWRKY26 gene and its homologous were constructed using TBtools, MEGA 12.0, and DNAMAN software. Following the construction of a GFP-VdWRKY26 expression vector, it was transformed into tobacco to determine the subcellular localization of VdWRKY26. The expression levels of VdWRKY26 were measured by qPCR in different tissues of V. davidii, and the promoter activity of VdWRKY26 was determined by GUS staining. Homologous expression in grape 41B suspension cells was used to validate its function.【Results】Pearson correlation analysis between VdWRKY26 gene expression levels and organic acid content revealed that VdWRKY26 exhibited high expression during the early stages of fruit development and low expression during the later stages, showing a significant positive correlation between VdWRKY26 gene expression levels with organic acid accumulation in V. davidii fruits. Sequence analysis indicated that the coding region of VdWRKY26 gene spans 1434 bp, encoding 477 amino acids. Phylogenetic analysis of VdWRKY26 and its homologous genes showed that VdWRKY26 belonged to the same clade as petunia PhPH3, pear PbWRKY26 and apple MdWRKY126. Using DNAMAN software to align the VdWRKY26 protein sequence with its homologous sequences from V. vinifera, apple, pear, tomato, Arabidopsis, Petunia, and Citrus, it was found that this protein contains a WRKY domain encompassing the β2, β3 and β4 regions. Subcellular localization analysis revealed that the GFP-VdWRKY26 protein was primarily localized in the nucleus. Subsequently, qPCR was used to detect the expression levels of VdWRKY26 in roots, stems, young leaves, fruits at 13 days after flowering (DAF), fruits at 49 DAF, fruits at 88 DAF, and fruits at 130 DAF. Results revealed that VdWRKY26 exhibited higher expression levels in leaves, fruits at 13 days after flowering (DAF), and fruits at 49 DAF, while showing relatively lower expression in roots and stems. This indicates that the gene may play a key regulatory role during the early stages of leaf and fruit development. Analysis of cis-acting elements in the VdWRKY26 promoter via the PlantCARE website predicted that this promoter primarily contains the light-responsive element Box4 and participates in drought-induced elements such as MBS. Subsequently, transient transfection was performed in tobacco plants followed by GUS staining analysis. Results showed distinct blue staining in leaves at the transfection site, while no staining was observed in the empty vector control group, confirming the activity of the cloned VdWRKY26 gene promoter. Homologous expression of the VdWRKY26 gene was performed in grape 41B suspension cells, using empty vector- transformed grape 41B cells as controls. The expression levels of the VdWRKY26 gene in the control group (EV) and VdWRKY26 transgenic grape 41B cells was detected by qPCR. Results showed that the expression level of VdWRKY26 in the transgenic grape 41B cells was only 32.135% of that in the empty vector transgenic control cells, indicating a co-suppression phenomenon. High- performance liquid chromatography was used to determine the organic acid content in EV and 35S:GFP-VdWRKY26 transgenic grape 41B cells. It was found that the malic acid content in 35S: GFP- VdWRKY26 transgenic grape 41B cells was significantly lower than that in the control group, while the citric acid content showed no significant changes. This confirmed that VdWRKY26 primarily participates in the regulation of malic acid accumulation in grapes.【Conclusion】This study cloned the CDS region and promoter sequence of the VdWRKY26 gene and analyzed its function. VdWRKY26 exhibits higher expression levels in leaves and fruits than that in roots and stems, showing high expression levels during the early stages of fruit development. The cloned 2545 bp promoter sequence of the VdWRKY26 gene exhibits promoter activity. The VdWRKY26 protein is localized in the cell nucleus and possesses the function of regulating malic acid accumulation in grapes.