Expression characteristics and transcriptional regulation analysis of VlCKX5 gene in grape
[Objective]Grapes(Vitis vinifera L.)are an economically important fruit crop in the world,and severe berry drop can affect grape yield and quality.The synthetic cytokinin analog N-(2-chloro-4-pyridyl)-N'-phenylurea(CPPU)is known to enhance berry set in grapes.Cytokinin oxidase/dehydroge-nase(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/dehydro-genase 5(VlCKX5)gene and its promoter were cloned,and bioinformatic analysis,expression specifici-ty and transcriptional regulation were performed to illustrate its role in grape berry setting.[Methods]In this study,we conducted experiments using Kyoho grapes(V.. vinifera L.× V..labrusca L.)as the ex-perimental material.The young berries were treated with 10 mg·L-1 of cytokinin-like growth regulator CPPU 5 days after anthesis.The treated berries were sampled at 1,2,4 and 8 days after treatment.Fur-thermore,at 13th day after anthesis,we systematically harvested roots,stems,leaves,inflorescences,tendrils and young berries from grapevines for subsequent tissue-specific analysis.The VlCKX5 gene re-gion 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.Fur-thermore,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,canes,leaves,in-florescences,tendrils and young berries under natural growth conditions,as well as in young berries fol-lowing treatment with the CPPU,were quantified using real-time quantitative reverse transcription poly-merase chain reaction(RT-qPCR).The activity of the VlCKX5 promoter was evaluated through histo-chemical staining with β-glucuronidase(GUS).To predict the transcriptional regulatory interactions in-volving 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 in-to 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 lo-calization 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 after CP-PU 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 YlHGold 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)interac-tion detection.A 1566 base pair segment of the VlCKX5 promoter,located in 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 encoded 546 amino acids.The molecular weight of VlCKX5 was 61.516 62 ku,the isoelectric point was 8.36,the instability index was 36.64,the fat co-efficient was 94.27,and the protein structure was stable.VlCKX5 had the closest homology to CKX amino acids in Chinese kiwifruit,and had a FAD domain and cytokinin binding site(CK-binding),be-longing to a typical CKX family.VlCKX5 was highly expressed in roots and leaves,followed by inflo-rescences,and the expression of VlCKX5 was significantly reduced at 1,2,4 and 8 d after CPPU treat-ment.Prediction of the cis-acting elements of the VlCKX5 promoter revealed elements containing hor-mones 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 in-volved in the transcriptional regulation of VlCKX5,and VlAGL6a was a key candidate transcription fac-tor for VlCKX5.Subcellular localization assay verified that VlAGL6a was localized in the nucleus.The results of fluorescence quantification showed that VlAGL6a was the highest in inflorescences,followed by berries and tendrils,and the lowest in roots,stems and leaves.The RT-qPCR results after CPPU treat-ment showed that the expression levels of VlAGL6a were significantly reduced on the 1st,2nd,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 was spe-cifically bound to the promoter of VlCKX5 gene and promoted the transcription of VlCKX5,which af-fected grape berry setting by regulating the level of cytokinin,which provides a theoretical basis for fur-ther analysis of the mechanism of grape berry set.
万方数据
维普
