The Mechanism Of Natural Astringency-loss In Chinese Pollination-constant And Non-astringent Persimmons

Pollination-constant and non-astringent (PCNA) fruit can be eaten like an apple without any postharvest treatment and is the most desirable for fresh consumption. So, it has been the main breeding objective. However, there are only a few cultivars of PCNA type, and they have very narrow genetic variability. The project of persimmon breeding is cornerd. Recentaly, a series of new PCNA types including'Luotian-tianshi' were found in Luotian country Hubei province. But it was not reported about genetic background and the value as parents for the breeding projects in persimmon. In this study, PCNA type origined in China was the main material, and non-PCNA and PCNA typesorigined in Japan were the materials as compared with Chinese PCNA types. The development pattern of tannin cell and the expreesion pattern of the genes related with tannin biosynthesis and tannin cogulation during the development of the fruit were studied, and its relationship with nature loss of astringency was analyzed. These studies would be provided scientific insights on the mechanism of the PCNA nature loss of astringency and genetic improvement in Japanese persimmon. The main results of this study are as follows:1. Tannin cell size in Chinese PCNA type was not distinctly increased after July and the biggest area was smaller than 30×103μm2; the accumulation of tannin continued until September, and some tannin were coagulated during the stage of accumulation. These results suggested development pattern of tannin in Chinese PCNA type between Japanese PCNA and non-PCNA type.2. The full length cDNA of LAR gene named DkLAR was cloned from the young fruit of'Luotian-tianshi'(Diospyros kaki Thunb., Chinese PCNA type) using degenerate PCR and Rapid amplication cDNA End (RACE) method, and registered in NCBI (GenBank accession number:EU747876). The 1356 bp long DkLAR cDNA contained a 1047 bp open reading frame (ORF) and encoded 349 amino acids. The DkLAR protein contained the THD, RFLP and ICCN motifs conserved in LAR protein, and include the Rossmann dinucleotide-binding domain. Expression pattern of DkLAR was coincident with the tannin cell development. 3. Four PDC cDNA fragments (named DkPDCl, DkPDC2, DkPDC3 and DkPDC4) and two full length cDNA of ADH gene (named DkADH1 and DkADH2) were cloned from the young fruit of 'Mopanshi' (Diospyros kaki Thunb., Chinese PCA type) using degenerate PCR, RACE and TAIL-PCR method. The expression patterns of DkPDC and DkADH were performed in the pulp of the fruit during the throughout of the fruit development. DkPDC2 expression level increased and DkADH1 expression level decrease at the late development stage of the fruit in which soluble tannin was converted into insoluble tannin. It suggested that the transcritive activity of DkPDC2 and DkADH1 was relatived with the PCNA nature loss astrigency.4. The fruits of the'Luotian-tianshi' (Diospyros kaki Thunb., Chinese PCNA type), 'xiaoguo-tianshi' (Diospyros kaki Thunb., Chinese PCNA type),'Suruga' (Diospyros kaki Thunb., Japanese PCNA type) and'Zenjimaru' (Diospyros kaki Thunb., Japanese non-PCNA type) were enclosed in polyethylene bags containing small amount ethanol on the tree to remove astringency. The astringency of the fruits from all materials except 'Suruga' disappered completely in 6 days, and the decrease of soluble tannin were correlated with a increase of insoluble tannin in treated fruits. The expressions of DkPDC and DkADH in peel, pulp, core and seed of the'Suruga' were low and not related with treatment of ethanol. The expression pattern of DkPDC and DkADH gene in 'Luotian-tianshi' was similar to in'Suruga', but after treatment, DkPDC2 and DkADHl expression level in seed was increased significantly. In 'Xiaoguo-tianshi' and 'Zenjimaru', the expreesion level of DkADH1 was very high, but decreased after treatment using ethanol; the DkPDC2 expression was high in seeds of the'Xiaoguo-tianshi' and 'Zenjimaru', after treatment using ethanol, increased in'Xiaoguo-tianshi' and changed little in'Zenjimaru'. DkADH2, DkPDCl, DkPDC3 and DkPDC4 expression level was low and no specific in pulp, peel, core and seed in 'Xiaoguo-tianshi' and 'Zenjimaru'. These results suggested that DkPDC2 and DkADHl gene in seeds played an important role in tannin congulation, and the order of ability of removing astringency during nature loss of astringency was'Zenjimaru'>'Xiaoguo-tianshi'>'Luotian-tianshi'.