Screening And Functional Identification Of The Key Transcription Factors Associated With Malate Biosynthesis In Wine Grape Cultivar Cabernet Sauvignon

The contents of organic acids in grape fruits is an important factor that affects wine quality and taste.The mainly organic acids in grape fruits are tartaric acid and malic acid.Tartaric acid does not participate in other metabolic pathways,while malic acid can be efficiently metabolized and greatly affected by external environmental.Thus study on the malate biosynthesis and metabolism and the related genes expression regulations are the main breakthrough to improve the contents of organic acids in grape.In this study,Cabernet Sauvignon,a grape variety with the largest cultivated area in Ningxia,was used as the material.Based on the transcriptome sequencing of four developmental stages from fruit formation to maturity（hard,green,veraison,maturity）and the organic acid contents determination from the same samples,the key transcription factors were screened out that specifically bind to the functional genes associated with malate biosynthesis and metabolism,in Cabernet Sauvignon fruits.Real-time fluorescence quantitative PCR was used to verify the mutual regulation of these key functional genes and their key transcription factors in different planting regions and different fruit developmental stages,as well as their comprehensive effects on the biosynthesis of organic acids such as malic acid.The binding sites and mutual transcriptional regulation between these transcription factors and their functional gene promoter regions were verified by electrophoretic mobility shift assay.The experimental work presented here provides one of the theoretical investigations and technical into cultivation of high-quality wine grapes in the future.The study results are as follows:（1）Based on the transcriptome sequencing results of Cabernet Sauvignon fruits,a total of 1973 transcription factors,which included 16 types of transcriptional binding domains and 22 binding patterns,were predicted that had strong interactions with 110 functional genes belong to 14 types of enzymes,i.e.,pyruvate kinase（PK）,pyruvate dehydrogenase（PDH）,phosphoenolpyruvate carboxylase（PEPC）,pyruvate carboxylase（PC）,citrate synthase（CS）,cis-aconitase（ACO）,isocitrate dehydrogenase（IDH）,ketoglutarate dehydrogenase（KGDH）,succinyl COA synthase（SCS）,succinate dehydrogenase（SDH）,fumarate carboxylic hydrase（FH）,malate dehydrogenase（MDH）,vacuolar membrane proton ATPase（VATPase）and vacuolar membrane proton pyrophosphorylase（V-PPase）associated with malate biosynthesis pathway.By the gene expression level and organic acid content of the same sample,differentially expressed genes and weighted gene co-expression network（WGCNA）were analyzed.Furthermore,by combining the external enzyme activities of the functional genes associated with malate biosynthesis,and the correlation between the functional genes and the transcription factors,as well as their domain type structures,finally,26 transcription factors corresponding to 9 key functional genes belonged to 5 types of enzymes（PEPC,PC,CS,FH and MDH）in malate biosynthesis of Cabernet Sauvignon fruits were identified.（2）Real-time fluorescence quantitative qRT-PCR was used to verify the mutual regulation of the above key functional genes and their key transcription factors in three different Cabernet Sauvignon planting regions and different fruit developmental stages.13 transcription factors corresponding to 6 functional genes belonged to 5 types of enzymes which are closely associated with organic acid such as malate were further selected.The correlation analysis showed that these transcription factors were significantly correlated with the organic acids related to the corresponding enzyme reaction process.（3）The electrophoretic mobility shift assay（EMSA）was used to verify the FH and MDH functional genes and their corresponding key transcription factors of which are most closely associated with malate biosynthesis in Cabernet Sauvignon fruits.The results showed that The FH functional gene VIT₁3s0019g03330 and its key transcription factor VIT₀ls0011g06200 had no binding.Another key transcription factor VIT₀8s0056g01230 had a specifically binds at 1666-1686 bp upstream of their functional gene promoter.The MDH functional gene VIT₁3s0019g05250 did not interact with its key transcription factor VIT₀6s0004g04960,while specific binding with another key transcription factor VIT₁0s0003g02070 occurred at 753-773 bp upstream of their functional gene promoter.