Molecular Cloning And Expression Analyses Of Tannin And Anthocyanin Transporting Gene In Persimmon

Proanthocyanidins (tannins) and anthocyanin are important flavonoid metabolites in plants. Although their precursors are generated in the cytoplasm, they are both formed and stored in the vacuole.Recent years, most of the structural genes and regulatory genes in proanthocyanidin and anthocyanin biosynthetic pathway have been cloned and thorough researched, the present research focus in their later transporting mechanism. In this work, PCNA type’Xiaoguo-tianshi’origined in China was the main material. PCNA type’Maekawa-jirou’origined in Japan and the non-PCNA’Mopanshi’were used as comparisons. We studied the development pattern of tannin cell as the fruit development. We also analyzed the expression of tannin transporting gene and its relationship with nature loss of astringency. Besides,we cloned anthocyanin transporting gene.We also analyzed the correlation between its expression and Persimmon Fruit Coloration.The aim is to provide a scientific basis for persimmon deastrigency mechanism research and genetic improvement. The main results of this work are as follows:l.The accumulation of tannin in C-PCNA continues until the late stage of fruit development(late September in Wuhan). Some soluble tannin transform into insoluble tannin during the accumulation. C-PCNA tannin cell is less than30×103μm2during fruit development that is the same as J-PCNA. J-PCNA tannin cell has a elongated form,but C-PCNA tannin cell is long. elliptic which is similar to elliptic form in non-PCNA. So the tannin cell development pattern in C-PCNA is different from J-PCNA and non-PCNA. As the C-PCNA loss its astringency naturally,"Dilution Effect" and "coagulation" act together.2.The full length cDNA sequence of DkMATEl gene was isolated from Diospyros kaki Thunb.’Xiaoguo-tianshi’. The1767bp DkMATEl cDNA sequence contains a1512bp ORF, encoding503amino acids. The deduced protein comprises two MatE domains and12transmembrane segments. In a phylo genetic analysis, DkMATE1clusters together with those identified proanthocyanidin transporting MATE proteins in other plant such as AtTT12and MtMATE1. Its expression correlates with tannin accumulation in Persimmon fruit expecially in C-PCNA. It suggests that DkMATEl can transports tannin monomers in Persimmon especially efficiently transports the unique component of tannin in C-PCNA. Besides, DkMATE1and DkANR have a very similar expression pattern. DkANR catalyzes the production of epicatechin from anthocyandin. AtTT12and MtMATE1Facilitate Vacuolar Uptake of Epicatechin3’-O-Glucoside for Proanthocyanidin Biosynthesis in Medicago truncatula and Arabidopsis. To infer, DkMATE1transports Epicatechin3’-O-Glucoside into vacuole in persimmon.3. Another MATE gene was cloned from Diospyros kaki Thunb.’Xiaoguo-tianshi’, which was named DkMATE2. Phylogenetic analysis indicates DkMATE2clusters together with those identified anthocyanin transporting MATE proteins in other plant. The expression level of DkMATE2increases gradually in all the three types of persimmon and the level in the peel of ripened fruit is much higher than that of young fruit.What’s more, the expression pattern of DkMATE2is very similar to the anthocyanin transporting MATE protein of grape. It suggests DkMATE2is relevant to anthocyanin accumulation in persimmon. Besides, it maybe transport the precursors of anthocyanin in persimmon.