Screening Of Genes Related To ABA Promoted Grape Anthocyanin Accumulation And Functional Analysis Of ABFs Transcription Factors

Present study using ‘Benibalado’ grape berries as experimental materials,to study the effect of ABA treatment on fruit quality and anthocyanin synthesis.The mechanism of ABA promoting grape coloring was explored by transcriptome sequencing.Transcription factors Vv ABFs were cloned and functional analyzed.The main results are as follows:1.In the early stage of véraison,grape clusters were treated with 300 mg/L ABA,water treated berries were used as control.The phenotypes of ABA treated and control berries were observed and anthocyanins were determined by UPLC-MS.Mechanism of ABA promoted anthocyanin accumulation was analyzed by transcriptome sequencing.The results shown that after 3 days of exogenous ABA treatment,grape berries were obviously colored,variety and content of anthocyanins were also increased.By KEGG enrichment analysis,we identified 52 DEGs that related to anthocyanin biosynthesis,modification and transportion,11 DEGs related to ABA signaling were also observed.MYB,b ZIP,NAC and other transcription factors that may regulate anthocyanin biosynthesis were alsoscreened from DEGs,the analysing of cis-acting elements in promoters showed that ABREs were identified in most of the promoters.The accuracy of RNA-seq was validated by q RT-PCR analysis of some candidate genes.Overall,ABA promote anthocyanin accumulation in grape is a complex process,including ABA signal transduction,anthocyanin biosynthesis,modification and transportion,fifteen transcription factors were likely involved in this biological process.2.The weight,titratable acid and total soluble solids of grape berries at different development stages were determined,meanwhile,we detected the concentrations of total anthocyanins and endogenous hormone ABA in grape skins.The expression of ABA signaling pathway and anthocyanin biosynthesis related genes in skins of different development stages were observed by q RT-PCR.Promoters of 6 anthocyanin biosynthesis related genes were cloned by PCR,the cis-acting elements in these promoters were predicted to exploring the mechanism of ABA promoted grape skin coloring and anthocyanin accumulation of ‘Benibalado’ grape at transcriptional regulation level.The results showed that: ABA treatment significantly increased the total soluble solid while decreased titratable acid of ‘Benibalado’ grape.ABA treatment significantly increased the anthocyanin content and endogenous ABA content in the skin of ‘Benibalado’ berries.After ABA treatment,the expression levels of 9 ABA signaling pathway genes and 6 anthocyanin biosynthesis related genes of ‘Benibalado’ grape skins were significantly increased.The results of cis-acting element analyzing showed that the promoter sequences of above six anthocyanin biosynthesis related genes contained several ABRE motif which were reported related to ABA signal response.In summary,our results suggested that 9 ABA signaling pathway genes may play an important role in grape skins coloring,two of them(Vv ABFs)may directly works with the promoter sequence of anthocyanin biosynthesis related genes,which are abundant of ABRE elements to positive regulate the transcription level of downstream genes and eventually modulate the skins coloring and anthocyanin accumulation of grape.3.ORFs length of Vv ABF1 and Vv ABF2 were 1344 bp and 1275 bp respectively,and the amino acids numbers of these two genes were 448 and 425 respectively.The results of phylogenetic tree analysing showed that Vv ABF1 and Lc ABF1(Litchi chinensis)were located in same subfamily,while Vv ABF2 was closed to At ABF1 in Arabidopsis.The subcellular localization results showed that both ABF coding proteins were located in the nucleus.q RT-PCR analysis of these two genes showed that Vv ABF1 was highest expressed in stem,while Vv ABF2 was mainly expressed in skins and pulp,and Vv ABF1 and Vv ABF2 were up regulated in the veraison stages of grape berries.The results of yeast one hybrid and double luciferase assays showed that ABFs could activate the activity of Vv MYBA1 promoter.Which suggested that ABFs play a positive role in the regulation of anthocyanin biosynthesis in grape berries.