Functional Characterization Of DkMYB15 In Proanthocyanidin Biosynthesis In Chineses Pcna Persimmon

Persimmon tannins are condensed tannins,and also known as proanthocyanidins(PAs).PAs are end products of flavonoid biosynthesis,and important metabolites widely found in many plants.Persimmon(Diospyros kaki Thunb.)is unique in accumulating a large amount of high molecular weight PAs in fruit flesh and cause strong astringency feeling.The biosynthesis of PA is including multiple structural genes and regulated by transcription factors.The MYB,b HLH and WD40 are the major transcription factors for PA regulation that form a complex of MBW,which was found involved in many plant species.In this study,Dk MYB15,the MYB transcription factor that was selected from transcriptome and proteome sequencing.The expression pattern of Dk MYB15 shows strong correlated with accumulation of the soluble tannins in Chinese pollination constant and non-astringent(C-PCNA)persimmon.To understand its role in PA biosynthesis in C-PCNA,the transient overexpression analysis was performed both in persimmon levels and fruit discs,then the PAs content and related genes expression was detected,and its targets gene and the interaction with Dkb HLH2 was analyzed.The results obtained in this study would provide a scientific basis for the creation of persimmon germplasm with high and low tannin content.The main results are as following.1.Dk MYB15 is an R2R3-MYB transcription factor with a total length of 633 bp and encodes 211 amino acids.Its C-terminus contains a conserved EAR inhibitory motif.In phylogenetic analysis,Dk MYB15 was grouped together with the previously reported PA repressors.The transcripts of Dk MYB15 showed highly expression in the middle and late stages of C-PCNA fruit development,but lower expressed in full fruit development stages of non-PCNA and J-PCNA fruit,which is negatively correlated with the accumulation of soluble tannins in C-PCNA persimmon.2.The results of subcellular localization showed that Dk MYB15 was localized in the nucleus,which indicates that Dk MYB15 function as transcription factor.Transient overexpression of Dk MYB15 in persimmon leaves and fruit discs both of them showed that soluble tannin was dramatically reduced.In addition,the expression of PA biosynthetic pathway related genes was also down-regulated,among them the CHS and ANR genes were much obvious.The yeast one-hybrid experiment shows that Dk MYB15 can bind to the ANR promoter.However,the CHS gene shows selfactivation and failed to validate whether Dk MYB15 could binds to its promoter or not.3.The Dkb HLH2 was screened in the persimmon transcriptome database and had a similar expression pattern with Dk MYB15 during fruit development stages.Based on yeast two-hybrid and bimolecular fluorescent complimentary assay,we found Dkb HLH2 can interacted with Dk MYB15.The PA biosynthesis-related genes downregulation after overexpression of either Dk MYB15 or co-overexpression with Dkb HLH2,and the soluble tannin also reduced.However,there was no significant difference between each other.In summary,Dk MYB15 is a repressive MYB transcription factor with a conservative EAR inhibitory motif.Dk MYB15 is highly expressed in later stage of CPCNA persimmon,which inhibited soluble tannins accumulation by repress the expression of PA biosynthesis pathway genes.This finding will contribute to clarify the scientific basis of PA accumulation in Chinese PCNA persimmon,and would aid for high and low tannin content germplasm invocation in future.