Isolation And Characterization Of ADH And PDC Genes Involved In Natural Loss Of Astringency In Chinese PCNA Persimmon

The loss of astringency naturally is a trait that is inherited and dominant in CPCNA cultivars but the natural de-astringency trait of JPCNA is qualitative and recessive. Therefore, CPCNA has great benefits for PCNA persimmon breeding comparing with JPCNA. Several aspects indicated that the astringency loss property of CPCNA cultivars is significantly different from the JPCNA types, which probably involves in coagulation of soluble tannins into insoluble tannins by acetaldehyde.The CPCNA types ‘Luotian-tianshi’ and ‘Eshi 1’ were the main material in this research, compared with JPCNA types ‘Maekawa-jirou’ and ‘Youhou’ and non-PCNA types ‘Mopanshi’. First, the key genes involved in the metabolism of acetaldehyde, alcohol dehydrogenase gene(ADH) and pyruvate decarboxylase gene(PDC), were isolated from CPCNA fruit. Then, expression of the genes were analyzed in persimmon using different astringent types(CPCNA, JPCNA and non-PCNA) at different development stages(5, 10, 15, 20 and 25 weeks after flowering) and different fruit tissues(flower, leaf, calyx, peel, pulp, seed and core). Combined with tannin, ethanol and acetaldehyde contents analysis, candidate genes that directly involved in tannin coagulation could be obtained. Finaly, the candidate genes were transiently transformed into persimmon leaves for function confirmation. These results will provide more evidences for elucidating molecular mechanism of natural loss of astringency in CPCNA cultivars. The main results of this research are as follows:1. in vivo transient transformation system with GFP as a reporter gene by Agrobacterium-mediated injection infiltration in persimmon leaves was established. Transformation conditions(concentration of acetosyringone, bacterial density and days after agroinfiltration) were optimized to achieve higher transformation efficiency. This system is suitable for different types of persimmon cultivars such as ‘Mopanshi’(PCA), ‘Eshi 1’(CPCNA) and ‘Baogai-tianshi’(CPCNA). Further, the developed transient transformation system was successfully utilized for the over-expression of Dk LAC1 gene in ‘Mopanshi’ and ‘Eshi 1’ persimmon leaves. The results exhibited that the Dk LAC1 expression leads to remarkably accumulation of proanthocyanidin(PAs) in persimmon leaves.2. A transient ihp RNA-induced gene silencing system based on Agrobacterium-mediated injection infiltration has been established in persimmon leaves in vivo. Dk PDS gene was chosen as a gene silencing target to evaluate the transient silencing system. Further, the developed transient silencing system was successfully utilized for Dk LAR gene silencing in ‘Mopanshi’, ‘Eshi 1’ and ‘Youhou’(JPCNA) persimmon leaves. DMACA staining and Folin-Ciocalteau assays showed that silencing of Dk LAR led to remarkably decrease in levels of PAs in persimmon leaves. This result indicates that Dk LAR gene involves in the PAs biosynthesis of persimmon.3. Based on Agrobacterium-mediated vacuum infiltration, a transient transformation system was established in tissue culture seedling of persimmon. GUS gene displays the highest transient transformation efficiency under the 20 min periods of vacuum. Further, the ihp RNA interference vector p HG-LAR targeted to Dk LAR gene was transferred to tissue culture seedling of ‘Mopanshi’ and ‘Xiaoguo-tianshi’(CPCNA) by using the developed transient transformation system. DMACA staining and Folin-Ciocalteau assays showed that PAs accumulation was remarkably inhibited in leaves of tissue culture seedling transformed with p HG-LAR vector. These results suggested that Dk LAR gene is required for PAs accumulation in persimmon.4. Tannin accumulation pattern of CPCNA types stopping until the late stages of fruit development is similar with the non-PCNA types. The levels of soluble tannins decreased rapidly at 20-25 WAF(weeks after flowering) in CPCNA, which led to natural astringency-loss of fruit. Furthermore, the levels of insoluble tannins increased rapidly during the later stages of fruit development(15-25 WAF) in CPCNA. These results indicated that the natural loss of astringency of CPCNA involves in tannins coagulation. However, soluble tannin contents of non-PCNA types remained at high levels even at maturity. The JPCNA types stop accumulating tannins at early stages of fruit development(10 WAF). The natural loss-astringency of JPCNA types is due to the dilution of soluble tannins by fruit growth.5. Three ADH genes(Dk ADH1, Dk ADH2 and Dk ADH3) and four PDC genes(Dk PDC1, Dk PDC2, Dk PDC3 and Dk PDC4) were isolated from the CPCNA fruit. The full length ORF of Dk ADH1, Dk ADH2 and Dk ADH3 is 1140 bp, 1137 bp and 1044 bp, respectively. The full length ORF of Dk PDC2 and Dk PDC3 is 1824 bp and 1743 bp, respectively. The partial c DNA sequences of Dk PDC1 and Dk PDC4 were obtained.6. The temporal and spatial expression analysis of ADH and PDC genes revealed that the Dk ADH1, Dk PDC1 and Dk PDC2 genes were up-regulated specifically during the late stages of CPCNA fruit development(20-25 WAF) in which the insoluble tannins contents remarkably increase. The expression levels of ADH and PDC genes were analyzed in different tissues of CPCNA types, with the highest expression levels were in seeds, especially Dk ADH1, Dk PDC1 and Dk PDC2 gene. These results suggest that Dk ADH1, Dk PDC1 and Dk PDC2 genes have close relationship with tannin coagulation of CPCNA.7. The ethanol and acetaldehyde contents in persimmon flesh and seed were measured at different developmental stages. The results displayed that the contents of both in seed are higher than that in flesh. The ethanol contents in seed increased rapidly at 5-15 WAF, and then declined remarkably at 15-25 WAF in both CPCNA and JPCNA types. The acetaldehyde contents in CPCNA seed gradually increased through fruit development, especially during the late stages of fruit development(15-25 WAF) the levels of acetaldehyde increased rapidly, which is consistent with the changes of insoluble tannin contents in CPCNA. The acetaldehyde contents in JPCNA seed showed fluctuation changes during fruit development. The ethanol contents in flesh showed a reducing trend through fruit development. The acetaldehyde contents in CPCNA and non-PCNA flesh gradually decreased accompanying with fruit development. However, it showed an opposite trend in JPCNA flesh.8. Dk ADH1 and Dk PDC2 genes were transiently transformed into ‘Mopanshi’ leaves for functional analysis. This result showed that the over-expression of Dk ADH1 and Dk PDC2 gene led to a significant decrease in levels of soluble tannin in persimmon leaves, respectively.In summary, Agrobacterium-mediated transient transformation system was established in persimmon leaves in vivo by injection infiltration and in tissue culture seedling of persimmon by vacuum infiltration, respectively. The mechanism of natural astringency loss of CPCNA types is probably related with the tannins coagulation. Dk ADH1, Dk PDC1 and Dk PDC2, are the key candidate genes for the coagulation of soluble tannins into insoluble tannins in CPCNA cultivar. This research provides gene resources and technical basis for further elucidating the mechanism of natural loss of astringency and breeding new CPCNA cultivar with independent intellectual property rights.