Bioinformatic Analysis and Hormone-Responsive Expression Profiling of CsDof3 and CsDof36 in Cucumber

Abstract

Background: Dof (DNA-binding with one finger) transcription factors are plant-specific zinc-finger proteins that regulate growth, development, hormone signalling, and stress responses. However, the structural features and hormone-response patterns of most cucumber Dof members remain unclear. Our previous RNA-seq screening identified two powdery mildew-responsive candidate genes, CsDof3 and CsDof36. Objective: To systematically characterise the molecular structures, tissue-specific expression patterns, and hormone responses of CsDof3 and CsDof36, thereby providing an important theoretical basis and genetic resources for further analysis of the molecular mechanisms by which they may regulate disease resistance through hormone signalling and for the molecular breeding of disease-resistant cucumber. Research Design: An approach combining bioinformatic prediction and gene-expression profiling was used to systematically characterise CsDof3 and CsDof36. Methods: ProtParam, ProtScale, SOPMA, PlantCARE, and STRING were used to predict the physicochemical properties, secondary structures, protein-interaction networks, chromosomal locations, and promoter cis-acting elements of the CsDof3 and CsDof36 genes and their encoded proteins. RT-qPCR was used to assess differences in the tissue expression of the two genes in cucumber roots, stems, cotyledons, and true leaves, as well as dynamic changes in transcript levels after treatment with SA, ABA, GA, and MeJA. Detailed concentrations and sampling times are provided in the Materials and Methods section. Results: CsDof3 and CsDof36 were predicted to be hydrophilic and unstable proteins, with random coils as their predominant secondary structures, and were located on chromosomes 1 and 6, respectively. Their promoters contained common MeJA- and GA-responsive cis-acting elements, whereas the CsDof3 promoter additionally contained ABA- and SA-responsive elements. CsDof3 expression was highest in cotyledons, whereas CsDof36 expression was highest in true leaves. Under hormone treatments, CsDof3 was consistently repressed by ABA, GA, and MeJA but was insensitive to SA. By contrast, CsDof36 was insensitive to MeJA, transiently repressed by SA, and induced by ABA and GA, indicating divergent regulatory response patterns.