Expression Analyses And Functional Confirmation Of Proanthocyanidin Precursor Transmenbrane Gene Mate In Persimmon Fruit

The persimmom(Diospyros kaki Thunb.) pulp contains specialized tannin cells, in which the large accumulation of proanthocyanidin (PA:persimmon tannin) is a major cause of astringent sense. As it was reported in many researches, Multidruge And Toxic Extrusion (MATE) transporter has been identified as candidate proteins for tannin precussor into the vacuole. However, its function in permission is unknown.In this work,’Eshi1’(C-PCNA), a pollination-constant non-astringent type persimmon that originated in China, was investigated in this research, compared with PCNA originated in Japan (J-PCNA,’Youhou’) and non-PCNA types (’Mopanshi’). The dynamic changes of insoluble and soluble PA content during the fruit development stages were studied. Expressing of DkMA TE1gene was analyzed by using different astringent stages (C-PCNA, J-PCNA and non-PCNA) at different development stages (5,10,15,20and25weeks after bloom), different fruit parts (flower, stem, leaf, calyx, peel, core, flesh). Moreover, Ectopic expression of DkMATE1in Arabidopsis thaliana tt12mutation and onion epidermal subcellular localization were employed to confirm its function. Furthermore, the correlation between the transported gene DkMATEl and nature loss of astringentcy was dicussed, then the molecule mechanism of nature loss of astringency in C-PCNA was proposed. The main results are as follows:1. The dynamic changes of PA content at different astringent stages:Soluble PA contents were at the highest level at5weeks after full bloom (WAB), and reduced with fruits development which is lowest at25WAB in three astringent stages.’Youhou’was natural de-astringent at10WAB (soluble PA content was lower than0.2%), however,’Eshi1’and ’Mopanshi’have not yet lost astringency.’Eshi1’was natural de-astringent at25WAB (mature before a week), but the soluble PA content of’Mopanshi’was still very high. The insoluble tannin contents of’Eshi1’increased at10WAB and had a peak at15WAB, while it decreased at the same time in’Youhou’and’Mopanshi’. It indicated that de-astringency of C-PCNA accompanied with the soluble tannin converting to insoluble tannin.2. Expression pattern of DkMATEl gene at different development stages, fruit parts and astringent stages:DkMATE1was expressed in flower, stem, leaf, calyx, core, peel and flesh of these three cultivars, and it showed highest expression in leaf which indicated that the DkMATEl gene is not specific expressed in fruits. The expression level of DkMATEl had a tendency to increase from10WAB to20WAB in’Eshi1’, while it decreased at the same time in ’Youhou’ and ’Mopanshi’. It can be inferred that DkMATEl gene might play a key role in the process of nature de-astringency in C-PCNA. The precursor selectively membrane-transported in DkMATEl is different between C-PCNA and J-PCNA. Maybe this is one of the reason of nature de-astringency in C-PCNA.3. The subcellular localization and functional confirmation of DkMATEl gene:By the method of Agrobacterium mediated transformation applied in Arabidopsis thaliana transparent testa12, the seed coat of which is light yellow because of lacking the TT2gene to concentrate tannin. T2generation seed of DkMATEl gene modified Arabidopsis ttl2recovered to wild-type of which the seed coat is yellowish-brown. It indicated mat DkMATEl could recover tt12mutation to wild-type with the function of transporting the proanthocyanidin precursor to vacuole. According to the analysis of onion epidermal subcellular localization mediated by gene gun, DkMATEl transport protein was located in tonoplast. The result was coincided with the function of transported tannin precussors.4. Substructure analysis of DkMATEl promoter:The1000bp of DkMATE1promoter sequence was obtained by Genomic Walking PCR method. Based on the analysis of promoter software, the data revealed that this sequence contains many cis-motifs of the MYB protein such as MYB1AT. MYBCORE、MYBPLANT、MYBST1. Moreover, MYC-BOX and W-BOX were existed in the promoter region. It provides evidence that the DkMATEl gene is regulated by the MYB transcript factors in the PA pathway.In summary, MATE, as a kind of transportprotein located in the vacuole membrane, encodes DkMATEl gene of which the rising expression level related with nature de-astringency in C-PCNA. It might have close relationship with selectiving membrane-transported the precursor of PA in DkMATE1, and it is worthy of further discussion.