Cloning Andcharacterization Of ALDH2 Genes Involved In Astringency Natural Loss In Persimmon

The mechanism of natural de-astringency in Chinese origin Pollination-constant and non-astringent persimmon(C-PCNA) is completely different from Japanese origin Pollination-constant and non-astringent persimmon(J-PCNA), which involves not only the effect of tannins cell ‘dilution’, but also the effect of soluble tannins “coagulation”. However, there are still no evidence about the relationship between de-astringency and key genes involving in the metabolism of acetaldehyde.In this work, based on transcriptome database of ‘Luotian Tianshi’(Diospyros kaki Thunb. C-PCNA), ‘Luotian Tianshi’(a pollination-constant non-astringent type persimmon that originated in China, C-PCNA),was applied and also compared with PCNA originated in Japan(J-PCNA, ‘Youhou’) and non-PCNA types(‘Mopanshi’). We studied the development pattern of tannin cell and the expreesion pattern of ALDH2 relating with tannin cogulation during the development of the fruit, and explore its relationship with natural de-astringency. This research will provide gene resources and techniccal basis for further elucidating the mechanism of the natural de-astringency in Chinese PCNA persimmon. The main results are as follows:1. In C-PCNA, tannin has been accumulating even in the late development period of the fruit with the conversion of soluble tannins into insoluble tannins. It indicated that the soluble tannins ‘cogulation’ was related to natural de-astringency in C-PCNA persimmon.2. Two genes of aldehyde dehydrogenase 2(ALDH2) gene family named Dk ALDH2 a and Dk ALDH2 b were obtained by screening and analyzing the transcriptome database of ‘Luotian Tianshi’. Then the full length c DNA sequences of candidate genes were obtained by using RACE and genomic walking methods. The 1563 bp long Dk ALDH2 a c DNA encodes 423 amino acids. The Dk ALDH2 a protein contains the ALDH-SF superfamily conserved motifs; the 1936 bp long Dk ALDH2 b c DNA encods 540 amino acids. The Dk ALDH2 b protein contains the ALDH-SF superfamily, ALDH_F2BC conserved domains and PLN02466 composite conserved domains.3. Expression of Dk ALDH2 aare detected in different organs of ‘Luotian Tianshi’, with the highest expression is in seeds. In ‘Mopanshi’, Dk ALDH2 a downregulated from 2.5 WAB to 20 WAB, while it increased suddenly at 25 WAB(Weeks after full bloom). The expression levels of Dk ALDH2 a in the ‘Youhou’(J-PCNA) is highest at 2.5 WAB, then downregulated accompanying with fruit development, which is consistent with the changes of soluble tannin content. In ‘Luotian Tianshi’(C-PCNA), the expression of Dk ALDH2 a decreased from 2.5 WAB to 20 WAB, while it increased suddenly at 10 WAB, then continued to decline accompanying with fruit development.4. Expression of Dk ALDH2 b were detected in different organs of ‘Luotian Tianshi’, with the highest expression was in core. The lower levels of Dk ALDH2 b in the ‘Youhou’(J-PCNA) are maintained. In ‘Mopanshi’, Dk ALDH2 b upregulated accompanying with fruit development, the fast increment is at 10 WAB. In ‘Luotian Tianshi’(C- PCNA), the expression of Dk ALDH2 b increased from 2.5 WAB to 5 WAB and decreased at 10 WAB, then continued to increase accompanying with fruit development.5. The astringency of the fruits disappeared completely in 6 days bytreating with ethanol, and the tendency of soluble tannin concentrationreduction was correlated with that of insoluble tannin concentrationincrement in treated fruits. In addition, the rapid decline of soluble tanninconcentration and increment of insoluble tanninconcentration in treated fruits were also observed corresponding to the tendency of Dk ALDH2 a expression level.In summary, natural de-astringency in C-PCNA persimmon may related to soluble tannins “dilution”. The expression patterns of Dk ALDH2 a and Dk ALDH2 b gene are associated with tannins coagulation, which indicated a key role of Dk ALDH2 a and Dk ALDH2 b in the metabolism of acetaldehyde.