| MiRNA and lncRNA play important roles in the reproduction of mammals and plants.However,there is no report about the regulation mechanism of miRNA/lncRNA involved in blueberry fruit development and maturation.Therefore,the research on the regulation mechanism of miRNA and lncRNA in the key differential genes of blueberry fruit ripening is an important way to explore the molecular mechanism of blueberry fruit ripening and color development,and it is also of great significance for blueberry molecular improvement and breeding.To reach this purpose,the high-throughput sequencing was conducted to identify the conserved miRNAs and predict their target genes at the blueberry mature stage,as well as further explore the role of miRNAs in regulating blueberry anthocyanin synthesis pathway.Analyses of the transcriptome for three typical stages of blueberry fruitswere performed to identify the differentially expressed genes(DEGs)and lncRNAs,and enriched the DEGs with related pathway,regulatory pathway analysis and metabolite analysis verification and other technical methods,predicted the interaction between lncRNA and mRNA were analyzed,and to obtain the mechanisms of miRNA and lncRNA regulating on blueberry fruit development and maturation.The main results are listed as follows:1.The mechanism of miRNA regulating in blueberry fruit anthocyanin synthesis.(1)In the present study,the genome-wide miRNA detection and functional analysis on the fruit of blueberry were conducted.Total of 20,909,994 accurate reads with a size of 18-30 nt,including 9,992,446 small RNA sequences were obtained in this study,indicating that blueberry fruits have a rich variety of small RNA types.The results of bioinformatics analysis on these miRNAs have revealed 412 conserved miRNAs belonging to 29 families,of which 6 families had more than 10 members,and 9 families had only 1 member.Through stem-loop qPCR,5 conservative miRNAs were randomly selected to verify their expression levels.In addition,35 new specifically expressed miRNA were identified in blueberries friut.(2)Furthermore,the potential target genes of conserved and novel miRNAs were predicted with psRNATarget online program and subjected to Gene Ontology(GO)annotation.Enrichment analysis of the GO-represented biological processes and molecular functions revealed that these target genes were potentially involved in a wide range of metabolic pathways and developmental processes.The target genes of conserved miRNAs appeared to be enriched in seed maturation,while those of novel miRNAs were significantly related to response to carbon dioxide,cellular ion homeostasis,zinc Ⅱ ion transport,regulation of nucleobase-containing compound metabolic process,and meiosis I.It is predicted that miRNA-regulated targeted functional genes involved in blueberry fruit anthocyanin synthesis indicate that multiple miRNA families can participate in anthocyanin synthesis with direct or indirect regulation mechnism.2.The mechanism of lncRNA regulating blueberry fruit ripening and development.(1)Through transcriptome analysis of blueberry young fruits(G group),middle fruits(W group)and matured fruits(P group),7,971 new genes were discovered,of which 3,389 were functionally annotated.27,140 differentially expressed genes(DEGs)were identified,and 16,058 lncRNAs were identified with a total of 2,215 differentially expressed lncRNAs.There are 10 genes were randomly selected to perfom qPCR and verify the relative expression of transcription.The results indicated their relative expressions were consistent with their FPKMs.(2)The results of genes’ function analysis have shown that the two pathways with the largest number of DEGs enriched in development-related pathways,the carbon metabolism pathway and the glycolysis pathway during the transition from G stage to W stage and then to P stage.Among them,the expression of D-3-phosphoglycerate dehydrogenase and serine O-acetyltransferase in the carbon metabolism pathway is significantly down-regulated and up-regulated in the G stage to W stage to control the cysteine synthesis,and the expression of glucose-6 phosphate isomerase,pyruvate decarboxylase,etc.in the glycolysis pathway were significantly increased.In the W stage to the P stage the synthesis of cysteine wasincreased by up-regulating the expression of D-3-phosphoglycerate dehydrogenase and serine O-acetyltransferase.glucose-6 phosphate isomerase and pyruvate decarboxylase also significantly increase the expression level to promote the glycolysis of blueberries.(3)The results of ripening-related DEGs analysis have indicated that plant hormone signal transduction pathways play an important role during the maturation process from G stage to W stage and to P stage.Among these key genes,the expression level of AHP from G stage to W stage is significantly increased to accelerate cell maturation and division,while the expression of the key gene for cell elongation TCH4 is significantly reduced,and both’s expression levels are reversed,that is,the expression level of AHP is significantly reduced.TCH4 is significantly increased.The ethylene receptor pathway related to fruit ripening is activated during W stage to P stage,and the key genes ETR,EIN3 and EBF1/2 all show significant up-regulation to promote blueberry fruit ripening development.(4)Through the targeted regulation analysis between lncRNA and mRNA of blueberry fruit,the number of cis and trans regulation pathway was 16051 and 16057,respectively.Further analysis revealed that there are 7 lncRNAs existed cis regulation function on the pathways of carbon metabolism,glycolysis and plant hormone signal transduction pathway,while there are more than 30 trans-regulated lncRNAs,indicating that the mechanism of lncRNA regulating blueberry fruit development and maturation has cis and trans regulation.The experiment of metabolome to verify the content of glycolytic anthocyanin-related molecules has showed that in the development process from G stage to W stage and to P stage,the differential metabolites include cysteine,pyruvate,citric acid,and sugar,their changes trends were consistent with the expression level of key genes in glycolysis.In summary,different types of ncRNA regulatory elements play different regulatory roles in the process of blueberry fruit anthocyanin synthesis,development and ripining.Among them,miRNA affects the synthesis of anthocyanins,a key component of blueberry fruit,by regulating the carbon dioxide response,intracellular ion balance,zinc Ⅱ ion transport,and the expression of base-containing complexes and other functional proteins.Research on the mechanism of lncRNA regulating blueberry fruit ripening found that it can regulate the expression of key genes in the carbon metabolism pathway,glycolysis pathway and plant hormone signal transduction pathway through the cis and trans mechanisms,and affect the proliferation and reproduction of blueberry fruit cells to achieve the maturation of blueberry fruit. |
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