Abstract 【Objective】Grapes (Vitis vinifera L.) are an economically important fruit crop in the world, and severe fruit drop can affect grape yield and quality. The synthetic cytokinin analog N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) is known to enhance fruit set in grapes. Cytokinin oxidase/dehydrogenase (CKX) enzymes, which are responsible for the irreversible degradation of
cytokinin, are pivotal in modulating plant growth and development. In the present investigation, the cytokinin oxidase/dehydrogenase 5(VlCKX5) gene and its promoter were cloned, and bioinformatic analysis, expression specificity and transcriptional regulation were performed to illustrate its role in grape fruit setting. 【Methods】In this study, we conducted experiments using
‘Kyoho’ grapes(Vitis vinifera L. × Vitis labrusca L.) as the experimental material. The young berries were treated with 10 mg·L-1 of the cytokinin-like growth regulator CPPU 5 days post-anthesis. The treated berries were sampled at 1,2,4 and 8 days post-treatment. Furthermore, at 13 days post-anthesis, we systematically harvested roots, stems, leaves, inflorescences, tendrils, and young fruits from plants for subsequent tissue-specific analysis. The VlCKX5 gene region was amplified via polymerase chain reaction (PCR). Bioinformatic analysis of the VlCKX5 protein sequence, including various physicochemical properties, was performed using the Expasy web tool. The identification of conserved domains within VlCKX5 was conducted through the InterPro database. Furthermore, the phylogenetic relationship among VlCKX5 and its homologs was examined using MEGA software Protein domain architecture of VlCKX5 and its orthologous proteins was examined utilizing the GeneDoc 2.7. Expression levels of VlCKX5 in grapevine tissues, including roots, stems, leaves, inflorescences, tendrils and young fruits under natural growth conditions, as well as in young fruits following treatment with the CPPU, were quantified using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The activity of the VlCKX5 promoter was evaluated through histochemical staining with β-glucuronidase (GUS). To predict the transcriptional regulatory interactions involving VlCKX5, we utilized the PlantTFDB, CIS-BP, and JASPAR databases to identify potential key transcription factors that may modulate its expression. The coding sequence (CDS) of VlAGL6, with the termination codon excised, was cloned into the 101LYFP vector. The construct was then transformed into Agrobacterium Competent Cells (GV3101), which were subsequently mixed with a selection marker and used to infiltrate Nicotiana benthamiana plants. At 72 hours post-transformation, the subcellular localization of fluorescence within N. benthamiana leaf cells was analyzed using laser scanning confocal microscopy. RT-qPCR was used to analyze the expression pattern of VlAGL6a in grape tissues and after CPPU treatment. A fragment of the VlCKX5 promoter containing the VlAGL6a binding site, designated as P, was cloned into the pAbAi vector, generating the recombinant bait plasmid pAbAi-proVlCKX5/P. This plasmid was then transfected into the Y1HGold yeast strain. Subsequently, the VlAGL6a gene was cloned into the pGADT7 vector to create the recombinant prey plasmid pGADT7-VlAGL6a, which was transfected into a yeast strain positive for the bait genome to perform yeast one-hybrid (Y1H) interaction detection. A 1566 base pair segment of the VlCKX5 promoter, located upstream of the ATG start codon, was cloned into the pGreenII0800-LUC vector to create a reporter construct. The VlAGL6a CDS was subcloned into the pSAK277 vector to produce an effector construct. Agrobacterium tumefaciens strains carrying these constructs were co-infiltrated into N. benthamiana leaves. The luciferase activity in the infiltrated samples was measured 48 hours post-infiltration using a dual-luciferase reporter assay system. 【Results】VlCKX5 was 1641 bp in length and encodes 546 amino acids. The molecular weight of VlCKX5 was 61.51662 kDa, the isoelectric point was 8.36, the instability index was 36.64, the fat coefficient was 94.27, and the protein structure was stable. VlCKX5 has the closest homology to CKX amino acids in Chinese kiwifruit, and has a FAD domain and cytokinin binding site (CK-binding) typical of the CKX family. VlCKX5 was highly expressed in roots and leaves, followed by inflorescences, and the expression of VlCKX5 was significantly reduced at 1, 2, 4 and 8 d after CPPU treatment. Prediction of the cis-acting elements of the VlCKX5 promoter revealed elements containing hormones responsive to IBA, SA and ABA. GUS chemical tissue staining test results showed that VlCKX5 activated its promoter activity in response to the treatment of CPPU, SA, IBA and ABA. Transcriptional regulation analysis showed that BPC, DOF, MADS and FLC family transcription factors may be involved in the transcriptional regulation of VlCKX5, and VlAGL6a is a key candidate transcription factor for VlCKX5. Subcellular localization assay verified that VlAGL6a localized in the nucleus. The results of fluorescence quantification showed that VlAGL6a was the highest in inflorescences, followed by fruits and tendrils, and lowest in roots, stems and leaves. The RT-qPCR results after CPPU treatment showed that the expression levels of VlAGL6a were significantly reduced on the 1th, 2th, 4th and 8th days, which was consistent with the expression pattern of VlCKX5. Y1H and double luciferase assay showed that VlAGL6a could interact with VlCKX5 and promote its expression. 【Conclusion】The VlCKX5 gene of grape responded to the signal of CPPU, and the transcription factor VlAGL6a specifically bound to the promoter of VlCKX5 gene and promoted the transcription of VlCKX5, which affects grape fruit setting by regulating the level of cytokinin, which provides a theoretical basis for further analysis of the mechanism of grape fruit set.
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