| Cerasus humilis (Bge) Sok and Cerasus japonica (Thunb.) are small shrub fruit trees which belongs to genus of cherry in Rosaceae. The both have similar tree canopy, but the organic acid contents in their fruits are different. Accumulation of the organic acids, activities of metabolism related enzymes and genes’ expression in Cerasus humilis (Bge) Sok and Cerasus japonica (Thunb.) were studied to figure out the difference in their organic acid metabolism. In this study, the titratable acid content was determined using acid-base titration while malic acid content and citric acid content were determined by high-performance liquid chromatography. The activities of NAD-MDH、NADP-ME、PEPC were determined. The conserved sequences of NAD-MDH, NADP-ME, PEPC and VHA genes in Cerasus japonica (Thunb.) were cloned by homologous cloning, and the expression of these genes during fruit development in Cerasus humilis (Bge) Sok and Cerasus japonica (Thunb.) were also studied. The main results are as follows:1.The content changes trend of titratable acid, malic acid and citric acid in Cerasus humilis (Bge) Sok were similar. In the early stage of fruit development, organic acid firstly went up slightly and then increased rapidly. The organic acid content decreased after the fruit was mature. The content changes of titratable acid, malic acid and citric acid in Cerasus japonica (Thunb.) were also similar. Except for the malic acid, all organic acids kept decreasing during fruit development. Malic acid was the major organic acid in Cerasus humilis (Bge) Sok and in Cerasus japonica (Thunb.) while citric acid was the second most in these two kinds of fruit. Malic acid and citric acid accounted for 85.34% and 10.98% of the total acid in the ripe fruits of Cerasus humilis (Bge) Sok, and accounted for 88.90% and 4.74% of the total acid in Cerasus japonica (Thunb.).2.The NAD-MDH, NADP-ME and PEPC were the key enzymes of malic acid metabolism in fruit. The NAD-MDH and PEPC were synthase, their activities were significantly positive correlated with the organic acid content in Cerasus humilis (Bge) Sok and Cerasus japonica (Thunb.). However, the correlation coefficient between PEPC’s activity and the organic acid content was lower than that of the NAD-MDH’s. The NADP-ME was degradative enzyme, the activities were significantly negative correlated with the organic acid content in Cerasus humilis (Bge) Sok and Cerasus japonica(Thunb.).3.The conserved areas of NAD-MDH, NADP-ME, PEPC and VHA genes in Cerasus japonica (Thunb.) were cloned, their length were 872bp,904bp,694bp,829bp respectively. These sequences shared more than 90% of similarity with the sequences from others species from the same family.4.The real-time PCR results showed that the NAD-MDH gene, NADP-ME gene and PEPC gene in Cerasus humilis (Bge) Sok (Ch) and Cerasus japonica (Thunb.) (Cj) had different expression patterns during fruit development, however after stone hardening stage these genes had similar expression patterns. The CjNADP-ME gene’s expression level was increased gradually in the early stage of fruit development while The ChNADP-ME gene’s expression level was decreased. The difference between the expression level of CjNADP-ME gene and ChNADP-ME gene may could explain the different accumulating patterns of the organic acid between the both species. The expression level of CjVHA gene was higher than ChVHA except the stone hardening stage and fruit enlargement stage. The expression levels of ChNAD-MDH gene and ChPEPC gene that boost the synthesis of malic acid were higher than that of ChNAD-MDH gene and ChPEPC gene,while the expression level of ChNADP-ME gene that promote the degradation of malic acid was lower than that of ChNADP-ME gene. However there was no correlation between each gene expression and organic acid content. |
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