Cloning of grape VvCCD1b gene and screening of transcription factors interacting with its promoter

【Objective】To screen transcription factors that interacted with the promoter of the grape carotenoid cleavage dixoygenase（CCD）gene VvCCD1b，and explore the potential factors that involved in the regulation of carotenoid metabolism of grape，which provided theoretical reference for further analysis in the metobolic regulation network of grape carotenoid.【Method】The cDNA and promoter sequences of the VvCCD1b gene were cloned. Bioinformatics softwares were utilized to predict and analyze the chromosomal location of the VvCCD1b gene，the cis-acting elements of the promoter，as well as the physicochemical properties and conserved domains of its encoded protein. Based on the transcriptome data，the expression patterns of grape CCD family members in different grape varieties and under different treatments were analyzed. Additionally，yeast one-hybrid technique was employed to screen for transcription factors that interated with the VvCCD1b gene promoter，and the point-to-point validation was performed.【Result】The 1641 bp full-length sequence of the VvCCD1b gene was obtained through PCR cloning，located on chromosome 13，encoding 546 amino acid residues. It was a hydrophilic protein with a secondary structure composed of α-helix（16.67%），β-fold（5.49%），extended chain（24.54%）and random coil（53.30%），and contained the RPE65 conserved domain. The VvCCD1b gene promoter contained transcription factors binding elements of light-responsive elements（G-box，3-AF1 binding site，GATA-motif， TCT-motif），salicylic acid（SA）responsive element SARE，and abscisic acid（ABA）responsive elements（ABRE）and AP2/ERF，WRKY，MADS-box. The expression level of the VvCCD1b gene gradually increased with fruit development and was affected by light and adversity stress. In addition，using the VvCCD1b gene promoter as bait，the transcription factors VvRAP2-4，VvWRKY4，VvbHLH137 and 18 functional proteins including grape vacuolar processing enzymes and glucosidases were preliminarily screened，among which the transcription factors VvRAP2-4 and VvWRKY4 interacted with the VvCCD1b gene promoter.【Conclusion】VvCCD1b is a hydrophilic protein，the gene expression level gradually increases with the fruit development，and is affected by drought，high temperature，waterlogging stress and light. Candidate transcription factors interacting with the VvCCD1b promoter are screened by yeast one-hybrid technology