Functional Characterization Of C2H2 Type Zinc Finger DkZF6 In Persimmon Proanthocyanidins Biosynthesis

Persimmon tannin(a kind of condensed tannin,also known as "proanthocyanidins",PAs)are widely exists in persimmon tannin cells.It is an important secondary metabolite in persimmon which can combine with human oral protein to produce "astringency" sensation in mouth.The biosynthesis of persimmon PAs is regulated by structural genes and transcription factor genes.At present,except the structural genes on PAs biosynthesis,most reports are focusing on the ternary protein complex mainly composed of MBW(MYB,b HLH and WD40).There are few reports on PAs biosynthesis regulation by other transcription factors.In this study,a Zinc finger protein might participate in PAs biosynthesiswas screened out from transcriptome and proteome database of persimmon.Phylogenetic analysis showed that this Zinc finger transcription factor clustered into C2H2 subfamily,named Dk ZF6.Based on dynamic expression variation determination,subcellular localization,transient transformation in fruit disc or leaves in persimmon,the function of Dk ZF6 was validated.The main results were as following.1.The full length of Dk ZF6 gene was 513 bp,encoding 170 amino acids,belonging to Q type C2H2 type Zinc finger protein.EAR inhibitory motif(Lx Lx L)exists at the Nterminal and the C-terminal.Subcellular localization assay of Dk ZF6 in tobacco showed that Dk ZF6 localized in the nucleus.Temporal and spatial expression pattern showed that Dk ZF6 was mainly expressed in persimmon fruit at 10 WAB.With the development of persimmon fruit,the expression level of Dk ZF6 increased,which was negatively correlated with the accumulation of soluble tannin in persimmon fruit.2.After overexpression of Dk ZF6 in ‘Mopanshi’ persimmon fruit discsand ‘Eshi 1’leaves,the contents of soluble tannin and total tannin decreased,together with down regulation of PAs pathway structural genes;opposite results were obtained after RNAi of Dk ZF6.The transient overexpression expression of Dk ZF6 cause down remarkable regulation of Dk CHS,Dk F3’5’H and Dk ANR.After heterologous expression in Arabidopsis,the seed coat color of whichwas slightly lighter than that of wild type after DMACA staining.3.Combined with yeast one hybrid experiment and double luciferase experimentrevealed that Dk ZF6 could interact with promoters of Dk ANR,Dk F3’5’H and Dk CHS-3 and repressed their activities during PAs biosynthesis.In conclusion,Dk ZF6 was negatively correlated with the accumulation of soluble tannin in persimmon fruit,which decreased the content of proanthocyanidins by inhibiting the activities of structural genes Dk ANR,Dk F3’5’H and Dk CHS.This study provides a new way to improve the regulation network of persimmon proanthocyanidins.