Characterization Of DkMATE7 In Transporting Proanthocyanidins Precursors In Chineses PCNA Persimmon

Proanthocyanidins(PAs)are important secondary metabolites affecting persimmon fruit quality.The biosynthesis and polymerization of PAs precursors take place in endoplasmic reticulum and vacuole respectively,so the precursors must PAs through the vacuole membrane to enter the vacuole.In this study,DkMATE7,a differentially expressed gene screened from C-PCNA ’Eshi 1’ transcriptome database,was selected as the research object.Through the study of its transmembrane transport mode in PAs biosynthesis pathway,the role of DkMATE7 in the process of PAs transmembrane transport was explored,the specific transport precursor was identified,and the regulation pathway of PAs metabolism in C-PCNA was further improved.The main results are as following1.DkMATE7 is 1461 bp in length and encodes 486 amino acids.It contains two highly conserved amino acid sequences CGQA and RVSNELGA related to PAs transport.The expression pattern of DkMATE7 in PCNA and non-PCNA was consistent with the accumulation pattern of soluble PAs content,but the expression level of DkMATE7 in PCNA was significantly higher than that in non-PCNA.2.DkMATE7 located on the vacuole membrane.After transient overexpression of DkMATE7 in persimmon pulp discs and living leaves,the content of PAs increased significantly,and the structural genes related to the PAs synthesis pathway were upregulated,while the content of PAs and the expression of related genes decreased after interference expression of DkMATE7.HPLC analysis showed that the PAs components of persimmon leaves expressing DkMATE7 were up-regulated The contents of Catechin(C),Epicatechin(EC)and Epicatechin gallate(ECG)increased significantly and decreased significantly after intervention,but the contents of Epigallocatechin(EGC)and Epigallocatechin gallate(EGCG)did not change significantly.3.The binding energy comparison of DkMATE7 and persimmon PAs five main subunits showed that the affinity of DkMATE7 with C,EC and ECG was significantly higher than that of EGC and EGCG;DkMATE7 transformed E.coli acr B mutant strains in the culture containing C,EC,ECG,EGC and EGCG respectively.Compared with the control,the DkMATE7 transformed mutant could only grow in the medium containing C,EC and ECG,which indicated that DkMATE7 had efflux activity of C,EC and ECG;Co-expression of DkMATE7 with DkLAR produced by catalyzing C and DkANR produced by catalyzing EC showed that DkMATE7 preferentially transports C,EC and ECG precursors in the process of PAs transport and accumulation.In conclusion,DkMATE7,a differentially expressed gene screened from ’Eshi 1’transcriptome database,was highly expressed in the early development of persimmon fruit and located on the vacuolar membrane.It preferentially transports Catechin,Epicatechin and Epicatechin gallate,the precursors of PAs,into the vacuole and participates in the accumulation of persimmon proanthocyanidins.The difference in the expression of DkMATE7 in PCNA and non-PCNA at the early stage of fruit development caused more proanthocyanidins precursors to accumulate in the vesicles of PCNA,which may be one of the reasons for the formation of PCNA and non-PCNA.The results further improved the regulatory network of proanthocyanidins metabolism in persimmon,and provided new ideas for molecular breeding and genetic improvement of the PCNA.