Study On Negative Regulation Of VvABCG20 Promoter Activity By Grape Transcription Factor VvDof14

VvABCG20 gene is higher expression in grape flowers and ovules,but lower in vegetative reproductive organs;there are differences in expression patterns between seeded grape and seedless grape during ovule development;the expression in seed coat is higher than that in endosperm;The homologous genes of VvABCG20 gene are involved in the biosynthesis of cork resin in seed coat and affects seed development,so we speculate that VvABCG20 gene can be used in seed development.It can participate in seed development by transporting seed coat related substances,and then affect ovule abortion.Promoters are involved in the regulation of downstream gene expression at the transcriptional level.Therefore,we study the promoter of VvABCG20 gene to explore the effect of promoters on the gene expression.In this paper,we cloned VvABCG20 gene in ’Pinot Noir’ and ’Thompson seedless’,constructed vectors of the VvABCG20 gene promoter and its deleted fragment that fusing body of GUS gene.The promoter activity of each fragment were analyzed by instantaneous transformation of tobacco leaves,and the promoter methylation and transcription factor regulation of VvABCG20 gene were analyzed.The expression of VvDof14 gene regulating VvABCG20 gene and its family were analyzed by ovule development and plant hormone treatment.The results are as follows:1 The VvABCG20 gene promoters of ’Thompson seedless’ and ’Pinot Noir’ were cloned and found to have two sequences with a full length and a deletion of 41 bp.The sequence was not significantly different between the two varieties.The full-length and deleted GUS fusion vector was constructed,and it was found by transient transformation of tobacco that the promoter lacked 5’UTR had no significant effect on promoter activity;the promoter was truncated to p586,p283,p197,p155,according to the position of the cis-elements.the promoter activity was sequentially decreased,and it was speculated that the cis-elements in the fragment were positively regulated to the promoter.GUS staining analysis of VvABCG20 gene promoter transgenic Arabidopsis thaliana showed that grape VvABCG20 gene was highly expressed in genital organs and embryos and low in vegetative organs.The CpG island region of the promoter region of VvABCG20 gene was amplified by BSP treatment of the DNA of ’Pinot Noir’ leaves and embryos.The methylation level was found to be stable in leaves and ovules by sequencing.Methylation was not caused by genes in tissues and organs.The reasons for the difference in expression,the specific reasons still need to continue to study.2 Using the website to predict the transcription factors that may interact with the VvABCG20 gene promoter,the VvDof14 gene was combined with the VvABCG20 gene promoter by yeast one-hybrid system,and the promoter was truncated to P1-P7 according to the number and position of Dof-motif.The VvDof14 gene binds only to the P7 fragment;the binding of VvDof14 to the VvABCG20 gene promoter and P7 is further confirmed by tobacco co-transformation experiments,and they are negatively regulated.The VvDof14 and GFP fusion expression vector were constructed to study the subcellular localization of grape VvDof14 protein.The VvDof14 protein was localized to the nucleus by confocal microscopy and was a typical transcription factor.The VvDof14-pGBKT7 vector was constructed and verified by transfection into yeast Y2 HGOLD strain.It was found that VvDof14 protein has no self-activating activity and can continue to interact with other proteins.3 The grape genome database and NCBI website identified 25 members of the grape Dof gene family.Through phylogenetic tree analysis,25 family genes can be divided into three categories,and the genes in each category have similar gene structures.Using ClustalX2 software to compare the protein sequences of 25 genes of grape Dof family,the similarity between the sequences of Dof family members was between 19.4% and 61%,and the highest similarity between AA160 and 220 was the Dof family gene.Conserved domain.RT-PCR analysis showed that the expression patterns of grape Dof family genes in ’Pinot Noir’ and ’Thompson seedless’ ovules were diverse,and most of the VvDof genes were highly expressed in the ovules of both varieties.Low,only VvDof10 expression in the ’Pinot Noir’ embryo is much higher than ’nuclear white’,and it shows the same pattern throughout the ovule development.The grape Dof family genes responded differently to different GA3,ETH,and MEJA plant growth regulators.VvDof6,VvDof14,VvDof20,and VvDof22 responded equally to the three hormones,and VvDof14 responded particularly to MEJA.