欧李、郁李有机酸代谢及相关基因表达的研究

发布时间：2019-02-15 00:35

【摘要】：欧李(Cerasus humilis (Bge) Sok)和郁李(Cerasus japonica (Thunb.))同属蔷薇科樱桃属的落叶小灌木果树,树形极其相似但果实有机酸含量差异较大。本文通过研究欧李及郁李果实有机酸积累、苹果酸代谢酶活性及其基因表达的差异,分析欧李和郁李果实中的有机酸代谢不同的原因。本文利用酸碱滴定法测定了欧李及郁李果实中可滴定酸的含量,用高效液相色谱法(HPLC)测定了苹果酸和柠檬酸的含量。测定了果实发育过程中欧李及郁李NAD-MDH、PEPC、NADP-ME酶活性的变化。采用同源克隆法从郁李果实中克隆了NAD-MDH、NADP-ME、PEPC、VHA基因的保守区序列,并分析了欧李及郁李果实发育过程中NAD-MDH、NADP-ME、PEPC、VHA基因的表达情况。主要结果如下：(1)欧李果实中可滴定酸、苹果酸和柠檬酸的变化趋势一致：果实发育前期,有机酸的含量不断增加,成熟前期迅速增加,成熟后有所下降。郁李果实中可滴定酸、苹果酸和柠檬酸的变化趋势基本一致：除接近成熟时苹果酸略有增加外,整个发育过程中的有机酸含量一直在降低。欧李和郁李果实中所含的有机酸最多的是苹果酸,其次是柠檬酸。成熟果实中,欧李的苹果酸含量和柠檬酸含量分别占总酸含量的85.34%、10.98%,郁李的苹果酸含量和柠檬酸含量分别占总酸含量的88.90%、4.74%。(2)NAD-MDH、PEPC和NADP-ME是苹果酸代谢的关键酶。NAD-MDH和PEPC主要促进了苹果酸的合成,两个酶的活性与欧李及郁李果实中苹果酸的含量都呈极显著正相关,但是欧李及郁李中PEPC酶活性与苹果酸含量的相关系数均低于NAD-MDH。NADP-ME的主要作用是促进苹果酸的降解,其酶的活性与欧李及郁李果实中苹果酸的含量都呈极显著负相关。(3)克隆获得郁李NAD-MDH、VHA、NADP-ME和PEPC四个基因的保守序列,长度分别为872bp、904bp、694bp、829bpo对克隆所得的各基因片段与其他近缘物种进行BLAST比对发现相似性都在90%以上。(4)荧光定量PCR结果表明,欧李和郁李果实中NAD-MDH、NADP-ME和PEPC基因的表达量变化趋势除在果实发育初期各不相同外,硬核期后基因的表达量的变化趋势基本相同。果实发育初期,郁李ME的基因表达量逐渐升高而欧李ME基因的表达量一直降低,使得两种果实的有机酸含量变化趋势不同。欧李VHA基因的表达量除在硬核期和膨大期略高于郁李外,其余发育时期都略低于郁李。在果实的整个发育过程中,促进苹果酸合成的NAD-MDH、PEPC基因在欧李中的表达量要高于郁李,而促进苹果酸降解的NADP-ME基因在欧李中的表达量低于郁李。但是各个基因的表达量和苹果酸含量之间并不存在相关性。
[Abstract]:Oli (Cerasus humilis (Bge) Sok) and Yu Li (Cerasus japonica (Thunb.) The tree shape of deciduous small shrub fruit trees of the same genus of Rosaceae Cherry genus is very similar but the content of organic acid of fruit is quite different. In this paper, we studied the difference of organic acid accumulation, malate metabolic enzyme activity and gene expression in the fruit of Euclidean and Yu plum, and analyzed the reasons for the difference of organic acid metabolism in the fruit. The content of titratable acid in the fruit of Eucalyptus chinensis was determined by acid-base titration, and the contents of malic acid and citric acid were determined by (HPLC). The changes of NAD-MDH,PEPC,NADP-ME activity during fruit development were measured. The conserved region of NAD-MDH,NADP-ME,PEPC,VHA gene was cloned by homologous cloning method and the expression of NAD-MDH,NADP-ME,PEPC,VHA gene during fruit development was analyzed. The main results are as follows: (1) the change trend of titratable acid malic acid and citric acid in the fruit of Euchamnus chinensis is the same: the content of organic acid in the early stage of fruit development increases rapidly in the early stage of ripening and then decreases after ripening. The change trend of titratable acid malic acid and citric acid in the fruit was basically the same: the content of organic acid in the whole development process was decreasing except for the slight increase of malic acid in the fruit near maturation. Malic acid and citric acid are the most organic acids in the fruit. In mature fruit, malic acid content and citric acid content accounted for 85.34% of total acid content, and malic acid content and citric acid content accounted for 88.90% of total acid content. (2) NAD-MDH,. PEPC and NADP-ME are the key enzymes of malic acid metabolism. NAD-MDH and PEPC mainly promote the synthesis of malic acid. However, the correlation coefficient between the activity of PEPC and malic acid content was lower than that of NAD-MDH.NADP-ME, and the main effect of NAD-MDH.NADP-ME was to promote the degradation of malic acid. The activity of the enzyme was negatively correlated with the malic acid content in the fruit. (3) the conserved sequences of NAD-MDH,VHA,NADP-ME and PEPC genes were obtained, the length of which were 872 BP, 904 BP, 694 BP, respectively. BLAST comparison of the cloned gene fragments with other related species by 829bpo showed that the similarity was more than 90%. (4) the results of fluorescence quantitative PCR showed that the NAD-MDH, in the fruit of Eulis and Eupatorium chinensis was similar to that of other relative species. The trend of expression of NADP-ME and PEPC genes was similar except in the early stage of fruit development, and the change trend of gene expression after hard nucleus stage was basically the same. In the early stage of fruit development, the expression of ME gene increased gradually, while the expression of ME gene decreased, which made the change trend of organic acid content in the two kinds of fruits different. The expression of VHA gene was slightly lower in the development period than that in the hard nucleus stage and the expansion stage, except in the hard nucleus stage and the expansion stage, and the expression level of the VHA gene was slightly lower than that in the Yu Li stage. During the whole development of fruit, the expression of NAD-MDH,PEPC gene which promoted malic acid synthesis was higher in European plum than that in Yu plum, while the expression of NADP-ME gene that promoted malic acid degradation was lower in European plum than that in European plum. However, there was no correlation between the amount of gene expression and malic acid content.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S662.5;S662.9

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