Cloning And Functional Identification Of The Key Genes Associated With Malate Biosynthesis In Wine Grape Cultivar Cabernet Sauvignon

In recent years,the contents of organic acids in grape(Vitis vinifera L.)fruits have been seriously insufficient due to global warming.The coloring and flavor of the fruits are also adversely affected,resulting in poor quality and taste of the brewed wine,which seriously affect the wine industry development and economic benefits.Malic acid is the most organic acid in grape fruit,which can be metabolized efficiently,and be greatly influenced by environmental factors.Therefore,it is particularly important to study the regulation of the related gene expression in malate biosynthesis and metabolism in wine grapes.In this study,Cabernet Sauvignon,the most cultivated grape cultivar in Ningxia,was used as materials.Its fruits at 4 developmental stages from formation to maturity were collected.The total RNAs were extracted for transcriptome analysis,and 8 kinds of organic acids closely related to malic acid were assayed.The key genes associated with malate biosynthesis in fruit were screened,then were cloned,and expressed in prokaryotic cells for enzymatic activity analysis in vitro.These key genes were verified by real-time fluorescence quantitative PCR(qPCR)using the fruits at the 4 developmental stages from 3 representative cultivation areas.Through the significance difference test and the correlation analysis between the level of gene expression and the contents of organic acids,the complex influences and the regulation mechanism in malate biosynthesis of these key genes in different cultivation area and developmental stage were comprehensively evaluated.The research provides a basis for genetic improvement of high-quality wine grapes.The research results are as follows:1.Through transcriptome analysis and determination of 8 organic acids(including phosphoenol-pyruvic acid,pyruvic acid,acetyl coenzyme A,oxaloacetic acid,citric acid,cis-aconitic acid,fumaric acid and malic acid)that closely related to malate biosynthesis in the fruits at 4 developmental stages(hard,green,turning and ripening),8 kinds of enzymes(including pyruvate kinase,PK;pyruvate dehydrogenase,PDH;phosphoenol pyruvate carboxylase,PEPC;pyruvate carboxylase,PC;citrate synthase,CS;aconitase,ACO;fumarate hydratase,FH and malate dehydrogenase,MDH),totally 16 functional key genes(including CsPK1,CsPK2,CsPDH1,CsPDH2,CsPEPC1,CsPEPC2,CsPC1,CsCS1,CsCS2,CsACO1,CsAC02,CsFHl,CsMDHl,CsMDH2,CsMDH3 and CsMDH4)associated with malate biosynthesis were screened by the correlation analysis between the level of gene expression and the contents of organic acids.Full-length of these key genes were cloned and sequenced.The specific gene sequences of Cabernet Sauvignon were identified and were found that had different mRNA transcripts,alternative splicing or SNP single nucleotide variation in each gene at different developmental stages of fruits,speculating that the transcripts regulated the biosynthesis and metabolism of organic acids during the fruit development.2.By constructing the prokaryotic expression vector(pTrc-CKS)which containing His-Taq tag,enzymatic activity analysis of these key genes were carried out in vitro.Except for the inability of CsPKl to express soluble protein,the other 15 genes had corresponding enzyme activities,but had significant differences.The enzyme activities of CsPK2,CsPDH2,CsPEPC2,CsCSl,CsACO1 and CsMDH3 were significantly higher than its homologous genes.It is speculated that these genes played more important role in the malate biosynthesis in Cabernet Sauvignon fruits.3.By selecting Cabernet Sauvignon fruits at the 4 developmental stages from 3 representative grape cultivation areas in East foot of Helan Mountain,the relative expression levels of each gene in each sample were analyzed by qPCR.The effects of fruit development stage and cultivation area on the expression levels and the organic acid contents of each key gene associated with malate biosynthesis in Cabernet Sauvignon fruits were verified.The expression levels of all key genes and the contents of most organic acids were approximate in different cultivation areas,but changed significantly during the fruit development.The gene expression levels of CsPK2,CsPEPC1,CsPEPC2,CsACO1,CsMDH2 and CsMDH4 varied greatly during different developmental stages of fruit;The organic acid contents,especially pyruvic acid and malic acid,also varied significantly,while acetyl-CoA,citric acid and fumaric acid varied smaller.4.Most of the key genes played a role in the biosynthesis of the corresponding organic acids.In particular,CsPEPC1 and CsPEPC2 catalyzed the formation of oxaloacetate from pyruvate,which in turn produced malic acid;CsMDH2 and CsMDH4 catalyzed the production of oxaloacetate from malic acid,while CsMDH2 and CsMDH4 did not catalyze the production of oxaloacetate but degraded malic acid,and CsACO1 catalyzed the formation of cis-aconitic acid from citric acid.These genes play a key role in the biosynthesis and metabolism of malic acid and citric acid in Cabernet Sauvignon fruits.