| The persimmon(Diospyros kaki Thunb.; 2n = 6x = 90) is the most economically important species within the genus Diospyros, which is believed to have originated in China where have the largest cultivator and producer worldwide. However, an overwhelming amount of persimmons cultivated in China are PCA type which need to remove the astringency before eating. Compared with the PCA persimmon, with the more commodity value of PCNA type is an important goal for the future persimmon breeding, because its astringency can be lost on the tree as the fruits develop and the fruit has a long shelf life. Previous studies have reported the C-PCNA trait is dominant over non-PCNA and J-PCNA, C-PCNA-type persimmon constitutes an important germplasm resource for PCNA cultivar breeding. In the recent study, astringency removal in C-PCNA persimmon is most likely related to soluble tannin coagulation, but the mechanisms of astringency removal in C-PCNA persimmon fruit are not well understood.In the present research, C-PCNA persimmon as the main material. Firstly, a c DNA-SSAP method was utilized to construct the c DNA library and to detect differentially expressed genes related to the “coagulation effect” during C-PCNA deastringency. Moreover, a further study on the mechanism of astringency removal in warm water will be conduct. Secondly, full-length complementary DNAs(c DNAs) of Dk PK genes were isolated from C-PCNA persimmon fruit based on a c DNA-sequence specific amplification polymorphism(SSAP) library by using the RACE and genome walking approaches. Furthermore, transient over-expression was used to confirm the function of candidate Dk PK genes in persimmon leaves. Lastly, yeast one-hybrid(Y1H) screen was performed in search of the transcription factors binding the Dk PK1 for figuring out the regulatory mechanism of astringency removal in PCNA cultivars. The study will be helpful for understanding the mechanism of soluble tannin coagulation and for the breeding of PCNA cultivars in the future. The main results of this research are as follows:1. c DNA library of C-PCNA persimmons treated with 40 °C water were constructed by a c DNA-SSAP approach to investigate the genes associated with deastringency, and q RT-PCR and transcriptome data confirmed the validity and accuracy of the c DNA library. The c DNA-SSAP analysis revealed that 283 differentially expressed transcript-derived fragments(TDFs) were successfully cloned and sequenced. These TDFs were involved in various molecular processes, including PA biosynthesis, transmembrane transport, pectin metabolism, and the solidification effect of PAs by using Blast2 GO analysis.2. The aldehyde-mediated coagulation effect and pectin production contribute considerably to persimmon deastringency in warm water. Notably, 3 aldehyde metabolism gene fragments and 1 pectin-related gene that are potentially involved in astringency removel were identified. The expression of those TDFs were all increased in the C-PCNA and non-PCNA persimmon fruit treated with 40 °C water. To our knowledge, this report is the first to identify these gene fragments in the persimmon. Combined with tannin and water soluble pectin(WSP) contents analysis, we proposed that the tannin-pectin complex formation and the aldehyde-mediated coagulation effect contributed to the deastringency of the persimmon fruit treated with 40 °C water.3. TDF 284-2(Dk PK gene fragment) potentially involved in the natural loss of astringency of C-PCNA cultivars were screened and identified from the c DNA-SSAP library. Furthermore, 6 full-length c DNAs of Dk PK genes were isolated from C-PCNA persimmon fruit by using the RACE and genome walking approaches.4. Dk PK1 may be involved in the natural removal of astringency in C-PCNA persimmon. The expression patterns of these 6 Dk PK genes and correlations with the soluble proanthocyanidin(PA) content were analyzed during various fruit development stages in different types of persimmon, with Dk PK1 showing an expression pattern during the last stage in C-PCNA persimmon that was positively correlated with a decrease in soluble PAs. Phylogenetic analysis revealed that Dk PK1 belongs to cytosolic-1 subgroup, and subcellular localization analysis confirmed that Dk PK1 is located in the cytosol. Furthermore, transient over-expression of Dk PK1 in persimmon leaves resulted in a significant decrease in the content of soluble PAs but a significant increase in the transcript levels of pyruvate decarboxylase genes(Dk PDC1, 3, 4, 5) as well as alcohol dehydrogenase genes(Dk ADH1, 3). Thus, we propose that an acetaldehyde-based coagulation effect reduces the content of soluble PAs. Taken together, our results suggest that Dk PK1 might be involved in the natural removal of astringency at the last developmental stage in C-PCNA persimmon. Finally, combining our present data with those from previous studies, a model is to be proposed for natural deastringency in C-PCNA persimmon.5. 4 upstream regulate factors interacting with Dk PK1 were isolated. About 2,000 bp of Dk PK1 promoter was isolated based on its full-length c DNAs sequence by genome walking. 140 monoclonal were isolated from yeast library based on the Yeast one hybrid screening, 4 upstream regulate factors including MADS-Box, WRKY, MYB and YABBY transcription factors were finally identified. In addition, 3 upstream regulate factors interacting with Dk CAD1 were isolated by using the same technique.In conclusion, a c DNA library incluing the 283 differentially expressed TDFs were constructed by a c DNA-SSAP approach. Combined with tannin and water soluble pectin(WSP) contents analysis in non-PCNA persimmon, we proposed that the tannin-pectin complex formation and the aldehyde-mediated coagulation effect contributed to the deastringency of the persimmon fruit treated with 40 °C water. In addition, 6 full-length c DNAs of Dk PK genes were isolated from C-PCNA persimmon fruit, and Dk PK1 potentially involved in the natural removal of astringency in C-PCNA persimmon was confirmed by transient over-expression system. Lastly, 4 upstream regulate factors incluing 3 novel proteins interacting with Dk PK1 were isolated from C-PCNA persimmon fruit by using the Y1 H screen. These successfully isolated genes and transcription factors will provide more scientific evidence for regulation of deastringency mechanism in C-PCNA and creation of PCNA new germplasm. |
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