Analysis of VlCKX4 Expression Characteristics and Prediction of Transcriptional Regulation in Grape

The cytokinin dehydrogenase/oxidase VlCKX4 and its promoter in grape (Vitis vinifera) were cloned to analyze the expression characteristics, promoter activity and transcription factor prediction, so as to provide a basis for the molecular mechanism that VlCKX4 involved in cytokinin-mediated berry set in grape.

The gene sequence of cytokinin oxidase/dehydrogenase 4 (VlCKX4) in Kyoho grape (Vitis vinifera × Vitis labrusca) was studied by bioinformatics method. The gene and its promoter were cloned by PCR. The expression characteristics of VlCKX4 were analyzed by real-time quantitative PCR (qRT-PCR). GUS activity assay was used to analyze the promoter activity. The transcriptional regulatory relationship of VlCKX4 was predicted by PlantTFDB, CisBP databases, and the output results were visualized by Gephi software.

The total length of VlCKX4 was 1 582 bp, which contained 1 566 bp open reading frame (ORF), encoded 522 amino acids, and had the family characteristic FAD domain and cytokinin binding (CK-binding) domain. qRT-PCR results showed that VlCKX4 was highly expressed in inflorescence and leaf, but the expression of tendril was lowest. The expression of VlCKX4 decreased first and then increased after treatment with cytokinin (CPPU), while the expression of VlCKX4 increased first and then decreased after treatment with the cytokinin inhibitor lovastatin (Lov). Cis-element analysis showed that the VlCKX4 promoter contained response elements of methyl jasmonate (MeJA) and other hormones. GUS histochemical staining showed that VlCKX4 responded to the hormones. Predictive analysis of transcriptional regulation showed that MYB, DOF and WRKY transcription factors were involved in transcriptional regulation of VlCKX4. Combined with transcriptomic expression data and coexpression relation, WRKY20, DOF1.5 and MYB59 were identified as key candidate transcription factors.

VlCKX4 was induced by CPPU and participates in the process of promoting grape berry set, during which prediction was regulated by transcription factors, such as WRKY20, DOF1.5 and MYB59.