Screening Transcription Factor Interacted With DkPDC2 In Persimmon By Yeast One Hybrid System

There are two kinds of natural deastringency mechanism of PCNA persimmons,called “dilution effect” and “coagulation effect”.The mechanism of natural deastringency in Japanese pollination-constant and non-astringent(J-PCNA)persimmon mainly caused by “dilution effect”,But in Chinese pollination-constant and non-astringent(CPCNA)persimmon both “dilution effect” and “coagulation effect”.The mechanism of natural deastringency in C-PCNA is directly related to the “coagulation effect”.Previous studies have confirmed that the key gene Dk PDC2 of acetaldehyde metabolism is closely related to “coagulation effect”,and acetaldehyde may mediate the conversion of soluble tannins into insoluble tannins,but the detailed mechanism is not defined.In this study,‘Luotian Tianshi'(C-PCNA)was used as experiment material,To obtain the transcription factors that cause the natural astringency loss of C-PCNA by regulating Dk PDC2,based on a high quality yeast hybrid c DNA library construction and interaction analysis.The main results of this research are as follows:1.The candidate transcription factor Dk MYB9 was obtained by screening the yeast library.Yeast one hybrid assay and Luciferase assay indicated that candidate transcription factor Dk MYB9 could activate Dk PDC2 promoter activity,implying that the interaction between Dk MYB9 and Dk PDC2.2.Phylogenetic analysis revealed that candidate transcription factor Dk MYB9 were grouped with At MYB3 and At MYB4,which inhibit anthocyanidins synthesis.The analysis of amino acid sequence revealed that R2R3 domain and MYB-b HLH binding sequence existed in the candidate transcription factor Dk MYB9.Transcripts level based on the expression level of Dk MYB9 and Dk PDC2 in various fruit development stages in different types of persimmon,suggested that the expression pattern of Dk MYB9 and Dk PDC2 genes was similar.3.Based on Agrobacterium-mediated vacuum infiltration in leaves of seeding in vitro,soluble PAs content decreased after overexpression of Dk MYB9.q RT-PCR analysis revealed that the proanthocyanidin biosynthesis pathway gene down regulated and the expression of acetaldehyde metabolism related genes was up-regulated.In conclusion,the candidate transcription factor Dk MYB9 leads to the reduction of soluble tannin content,which also resulted in the up-expression of acetaldehyde metabolism gene,further confirmed that the “coagulation effect” is closely related to the natural astringency of C-PCNA.