Transcriptional Regulation Analysis Related To Sugar Content During Fruit Development And Functional Analysis Of Sugar Transporter MdTST In Apple

Fruit sweetness is one of the most important indexes to measure fruit quality,which mainly depends on the accumulation of sugar in the fruit.Fruit sugar content is highly regulated by metabolism and transportation.In order to explore the transcriptional network regulated by sugar content during apple fruit development,this dissertation used ATAC-seq technology combined with RNA-seq data to identify the key genes and upstream transcription factors(TF)related to sugar/acid metabolism and transportation during ‘Fuji’apple fruit development.The function of MdTST,a vacuolar membrane sugar transporter closely related to fruit sugar accumulation,was verified,which revealed that MdTST1/2 play key roles in apple fruit sugar accumulation,and the function of MdCBF1/2,two candidate transcription factors of MdTST1/2,in regulating fruit sugar accumulation at low temperature were analysed.The main research results are as follows:1.Transcriptional network analysis regulated by sugar content during apple fruit development.The transcriptional levels of key genes involved in sugar/organic acid metabolism and various sugar transporter genes were analyzed in mature leaves and eight fruit samples of ‘Fuji’ apple.The results showed that the expression level of genes such as TST、ERDL6、SOT、SPS and ALMT were closely related to the accumulation of sugar acid during fruit development.By weighted gene co-expression network analysis(WGCNA),2,6 and 69 TFs was identifyied associated with the accumulation of glucose(Glc)/ fructose(Fru),sucrose(Suc)and malate / sorbitol,respectively.The ATAC-seq technique was further used and enrich 502 motifs at young fruit stage,expansion stage and maturity stage.The results of ATAC-seq and RNA-seq data were then analyzed by association analysis,and an average of 686,649 pairs of potential TF-target genes were obtained by scanning the differentially expressed gene promoter regions and differentially open regions in each differential group.The results showed that OBP1/3,BPC1/5/6,ERF2/9,FLC,SVP,TCX2,PI,SOC1,CBF1/2 and other motifs were enriched in the promoter regions of 305 genes related to sugar/acid metabolism.Luciferase reporter assay and VIGS-mediated gene silencing technique were used to verify the interaction between TFs such as BPC1,OBP3 and the promoters of selected target genes.The results indicated that these TFs may affect sugar accumulation by regulating downstream target genes related to sugar accumulation.In addition,a TF-TF regulatory network was constructed based on the fact that transcription factors that simultaneously act on the same differentially expressed target genes in differentially open regions may have the tendency of co-regulation.2.Functional verification of apple sugar transporter MdTST in fruit sugar accumulation.Six members of MdTST genes family,MdTST1-6,were identified in apple genome GDDH13V1.1.The expression characteristics showed MdTST1 and MdTST2 were with the highest expression abundance in fruits,and the expression trend during development was consistent with fruit sugar accumulation.The subcellular localization results showed that MdTST1 and MdTST2 localized to the vacuolar membrane.The results of sugar feeding experiment showed that MdTST1/2 promoter could sense Glc,Fru and Suc sugar signals and induce its own expression.Two overexpression lines were obtained by heterologous overexpression of MdTST1/2 in wild-type and At TMT1/2 double mutant tmt1-2::t DNA Arabidopsis,respectively.The results showed that overexpression of MdTST2 in the double silenced lines normalized the growth-deficient phenotype.Sugar content analysis showed that under the background of tmt1-2,overexpression of MdTST1 had no obvious effect on the contents of three kinds of soluble sugars,while the concentrations of Glc,Fru and Suc in MdTST2 overexpression lines increased significantly and returned to the level close to the wild-type.In addition,after overexpressing MdTST1 and MdTST2 in wild-type plants,the sugar content was both significantly increased compared with that of wild-type plants.In addition,after heterologous expression of MdTST1 and MdTST2 in ‘Micro-Tom’ tomato,the contents of Glc,Fru and Suc in the fruit of transgenic tomato lines were significantly increased,indicating that MdTST1 and MdTST2 fruits play important roles in the accumulation of sugar.3.Transcription factors MdCBF1/2 regulate the expression of MdTST1/2 and sugar accumulation at low temperature.Combined with ATAC-seq data and MdTST1/2 promoter cis-acting element analysis results,it was supposed that the transcription factor MdCBF may regulate the expression of MdTST1/2.After low temperature treatment,the contents of Glc,Fru and Suc in apple leaves and fruits increased significantly.q RT-PCR analysis showed that the expression levels of MdTST1/2 and MdCBF1/2 were significantly up-regulated.Ch IP-q PCR,luciferase reporter assay,GUS activity detection and yeast monohybrid assay showed that MdCBF1/2 could bind to the MdTST1/2 promoter regions and promote the expression of MdTST1/2.Furthermore,the overexpression and silencing vectors of MdCBF1/2 were constructed and transform into the calli of ‘Orin’ apple.In the apple calli lines with overexpressing MdCBF1 and MdCBF2,the transcription levels of MdTST1 and MdTST2 were significantly up-regulated,and the content of soluble sugar was significantly increased,especially the content of Glc was increased by more than 2 times.However,in the silenced calli lines of MdCBF1 and MdCBF2,the transcription levels of MdTST1 and MdTST2 were significantly inhibited,and the contents of Glc,Fru and Suc were decreased.In addition,silencing MdTST1/2 by VIGS in the transgenic calli with overexpressing MdCBF1/2 showed that the soluble sugar content decreased significantly,especially the sugar content decreased most after silencing MdTST2.These results indicated that the increase of sugar content in MdCBF1/2 transgenic callus was related to the expression of MdTST1/2.