The Possible Mechanism Of Dna Methylation Regulates Anthocyanin Accumulation Of Blueberry(V.Corymbosum)

DNA methylation is an important epigenetic mechanism,which regulate the expression of numerous vital genes while its base compositions have not been changed,Plants mainly contain four cytosine methyl transferases:the METs family of DNA methyltransferases,the domain-recombinant methylase DRMs,the chromatin methylase CMTs and the DNA methyltransferase Dnmt2 family.Anthocyanins are the main metabolites in blueberries and other fruits and vegetables,and have important nutrition.At present,the relative researches on blueberry anthocyanins mainly focused on the regulation and metabolism of synthetic pathways and extraction methods improvement,etc.The effects of DNA methylation and demethylation genes regulating anthocyanin biosynthesis is rarely known.Therefore,the aim of this paper was to evaluate the possible mechanisms of DNA methylation and demethylation regulating blueberry anthocyanins accumulation.The related genes of DNA methylation and demethylation and anthocyanin biosynthesis from Southern highbush blueberry ’O’Neal’ and ’Bluerain’ were isolated.The relative expression levels of DNA methylation and demethylation genes,the anthocyanin content,and the nucleotide structures of anthocyanin biosynthetic gene promoter were analyzed.The results were as follows:(1)VcMET1,VcDRM2,VcCMT3 and demethylation gene VcDME1 were isolated from blueberry respectively.VcMET1 contained two DNMT1-RFD domains,two N-terminal regulatory regions,transcriptional regulation domain BAH and one cytosine methyltransferase Dcm domain.VcDRM2 contained one ubiquitin related domain UBA and one S-adenosylmethionine(AdoMet)dependent methyltransferase domain.VcCMT3 contained one BAH,Dcm and chromatin structure modification domain CHROMO,respectively.VcDMEl contained one RRM_DME,one HHH super family encoding helical hairpin structure,and one Perm-CXXC protein domain detectable by Demeter-like protein.Moreover,the expression levels of VcMETl and VcCMT3 gradually decreased during the fruit development.whereas VcDRM2 mRNAs presend a trend of declining during the whole fruit development except stages S6 and S7.(2)VcANS,VcDFR,VcCHS,VcF3H and VcUFGT,the key genes regulating anthocyanin biosynthetic pathway,were isolated,and its expression patterns during the developmental stages were analyzed.The results showed that these genes exhibited a highly consistent dynamic trend of rising-falling-rising-decreasing.(3)Blueberries began to accumulate anthocyanins in the peel and from stage S5,then the anthocyanin content increased rapidly.The fruit at stage S5 exhibited white or light green,then switched to red or purple,till to a dark blue at stage S6 finally.The anthocyanin content were 0.005,0.22,2.63 mg/g.FW in the stage S5,Br and S6 fruits,respectively.Our results indicated that the high-level expression of anthocyanin biosyntheticgenes could improve anthocyanin accumulation in blueberry fruits,and the anthocyanin accumulation provided negative regulation the expression of anthocyanin biosynthestic genes.(4)The promoter sequences of VcAINS,VcDFR,VcCHS,VcF3H and VcUFGT,were isolated from ’O’Neal’ and ’Bluerain’.Online PlantCARE software predicted that CAAT-box and TATA-box were the most abundant functional sites,,and the light-responsive elements was about 40.30%,indicating that light might significantly influence on the anthocyanin biosynthesis.(5)Methylation level analysis of VcANS and VcCHS promoter sequences revealed that 2,10 and 8 cytosine sites were changed in the VcANS promoter from stage S5 to S6.And the cytosine modification of VcANS promoter was found in three major transcriptional start sites(two TATA-boxes and one CAAT-box)before and after breaking stage.As to VcCfHS promoter,2,2 and 4 cytosine sites were changed from stage S5 to S6,and happened in the transcription start site.Similarly,DNA modification at the CAAT-box and 5’-UTR Py-rich stretch region of VcAANS and VcCHS promoter affect the transcription and expression of the anthocyanin biosynthetic related genes.