Cloning And Function Characterization Of Proanthocyanidin Precursor Transporting Gene DkAHA1 In Persimmon

Proanthocyanidins（PAs）is a compound in persimmon,which cause the astringent sensation in mouth.In plant cells,PAs are synthesized in the cytoplasm and accumulated into the vacuole and apoplast.Tonoplast vacuole membrane P-ATPase TT13（AHA10,autoinhibited H⁺-ATPase isoform 10）can produce the driving force of proanthocyanidin precursor transport into the vacuole of Arabidopsis thaliana.While the function of AHA10 has not yet been elucidated in persimmon.The representative variety of Chinese sweet persimmon‘Eshi1’was used for isolating AHA10homologues based on persimmon transcriptome dataset.The sequence of DkAHA1was analyzed by bioinformatics and was characterized by transient over-expression or RNAi of DkAHA1 in persimmon fruit disc.The main results are as following:1.There are five homologue persimmon AHA,among which DkAHA1 encoded ORF of 2853bp.Phylogenetic analysis showed conservative DkAHA1 belong to P_3Atype ATPase.2.DkAHA1 was highly expressed in persimmon leaves and sepals,DkAHA2was mainly expressed in the core of fruit,DkAHA3 and DkAHA4 were mainly expressed in leaves,and DkAHA5 was mainly expressed in sepal.The expression of DkAHA1 exhibited similar variation tendany at different stages of fruit development in three persimmon varieties which was according to PAs content changes.3.Subcellular localization showed that the green fluorescence of DkAHA1protein coincided with the red fluorescence of tonoplast,this indicated that DkAHA1are distributed on the tonoplast.The content of proanthocyanidins decreased and increased correspondingly in persimmon slices with interference expression and over-expression of DkAHA1.4.The 1132bp promoter sequence of DkAHA1 gene was obtained from‘Eshi1’.There are 17 cis elements on the promoter which respond to environmental changes.The bait strain Y1HGold[p AHA1-Ab Ai]was constructed for the screening of related regulatory genes.The lowest inhibitory concentration of reporter gene Ab A^rbackground expression was 600 ng/m L.Above all,the expression pattern of DkAHA1 is same as the accumulation pattern of procyanidine in persimmon,which may participate in the accumulation process of procyanidine;as tonoplast protein,DkAHA1 can promote the accumulation of PAs,which may be influenced by the PAs precursor transport across the membrane.