| Proanthocyanidins(PAs,also known as condensed tannins)are abundantly present in the vacuole of the “tannin cell” specialized in the flesh of persimmon.Tannin can cause the sensation of astringency due to coagulation of oral proteins.The precursors of PAs have different sites of generation,polymerizaton and storage.The generation of PAs occurs in the cytosol,while its polymerizaton and storage occurs in vacuoles.GST is involved in the transport of anthocyanins and procyanidins precursor into the vacuole in Arabidopsis and Vitis vinifera,the function of GST gene for transporting PAs in persimmon is not clear yet.In this work,Chinese PCNA ‘Eshi 1’,Japanese PCNA ‘Youhou’ and non-PCNA ‘Mopanshi’ was used as material to investigate the role of DkGST1 gene in the transmembrane transport of PAs.The results provide a scientific basis for elucidating mechanism of PAs metabolic regulation in persimmon.The main results are as following.1.The content of soluble PAs in three astringent types of persimmon differs greatly,and the general trend is decreasing during fruit development.J-PCNA type reached 0.2% after 8 WAB,and the taste no obvious sensation of astringency.C-PCNA type reach 0.2% until about 25 WAB,the concentration of insoluble PAs showed an slightly increasing after 15 WAB.The content of soluble PAs in non-PCNA types remains at about 0.6% after 27.5 WAB,and it need artificial astringent treatment for consumption.2.The transcripts level of DkGST1 presented highest in sepals,followed by the leaves.The expression pattern of DkGST1 was quite different in C-PCNA type and J-PCNA type.During the development of J-PCNA type,the expression level of DkGST1 decreased continuously,accompany with the content of soluble and insoluble PAs decreasing.The expression level of DkGST1 show progressively increasing after 10 WAB in C-PCNA type,together with the soluble PAs concentration reduction.3.Gene overexpression or silencing of DkGST1 can significantly increase or reducing the content of soluble PAs by using Agrobacterium-mediated genetic transformation in persimmon leaves.The results of subcellular localization showed that DkGST1 located in the tonoplast of tobacco mesophyll cells,which was consistent with its transport function.4.The 986 bp of DkGST1 promoter sequence was isolated by Genomic Walking PCR method,it contained several cis-acting elements that could bind to MYB,WRKY,and MYC transcription factors,indicating that this promoter may be regulated by MYB and other transcription factors.The GUS activity analysis of the promoter showed that the obtained promoter contains transcription initiation site.In summary,DkGST1 differs in the expression patterns of C-PCNA and J-PCNA during fruit development,which may be related to their different PAs metabolism mechanisms.The GST encoded by this gene acts as a tonoplast protein can promotes the accumulation of PAs,probably related to its transmembrane transport of PAs precursors. |
