枣果实发育过程中主要有机酸含量变化及其相关代谢的研究

发布时间：2018-01-25 10:14

本文关键词： 枣有机酸酶活性基因表达　出处：《塔里木大学》2017年硕士论文　论文类型：学位论文

【摘要】：本研究以骏枣和酸枣为试材,采用高效液相色谱-二极管阵列检测器法(HPLC-DAD)对枣果实发育过程中有机酸组分与含量进行了检测,同时利用分光光度发对(苹果酸脱氢酶(NAD-MDH)、磷酸烯醇式丙酮酸羧化酶(PEPC)、苹果酸酶(NADP-ME)、柠檬酸合成酶(CS))等代谢酶活性进行了检测,以及通过qRT-PCR对枣果实发育过程中的主要有机酸相关调控基因的相对表达量进行了动态检测分析。结果表明:(1)建立了枣果实中有机酸含量的HPLC测定方法,7种有机酸组分和Vc标准曲线的(R2)均在0.998 9以上;精密度检测出其相对标准偏差为0.98%~4.77%(n=5);重复性检测结果显示器相对标准偏差值为0.88%~2.19%(n=5);回收率均在91.35%~105.74%之间;得出在成熟期枣果实中的有机酸含量由高到低顺序依次为:苹果酸柠檬酸酒石酸奎宁酸草酸富马酸,其中苹果酸含量最高可达19.21 mg·g-1,占同期总酸含量的89.76%;在果实发育过程中,除酒石酸外,苹果酸和柠檬酸含量的变化均呈先升高后降低的趋势。(2)苹果酸脱氢酶(NAD-MDH)和柠檬酸合成酶(CS)活性变化与苹果酸和柠檬酸含量的变化趋势基本一致,均呈现伴随果实发育而上升,伴随果实成熟而降低的趋势,苹果酸酶(NADP-ME)活性变化则相反。相关性分析表明:NAD-MDH活性变化与苹果酸含量变化呈正相关,NADP-ME活性变化与苹果酸含量变化呈负相关;CS活性变化与柠檬酸含量达到极显著正相关,相关系数可达0.9260;而磷酸烯醇式丙酮酸羧化酶(PEPC)活性变化与苹果酸和柠檬酸含量相关性不大。本研究认为:NAD-MDH、NADP-ME和CS活性对枣果实中有机酸代谢发挥重要作用,而PEPC作用不大。(3)在枣果实发育过程中,柠檬酸合成酶基因(ZjCS)的相对相对表达量的变化趋势与柠檬酸含量的变化趋势基本一致,呈先上升后降低的趋势,柠檬酸的积累主要受ZjCS基因的调控作用。而苹果酸脱氢酶基因(ZjMdh)、磷酸烯醇式丙酮酸羧化酶基因(ZjPEPC)和苹果酸酶基因(ZjMe)的表达模式在酸枣和骏枣中则表现不一,在酸枣果实中,苹果酸的积累主要受到NAD-MDH酶催化同时受到ZjMdh基因的调控作用。而在骏枣果实中苹果酸代谢的ZjMdh、ZjPEPC和ZjMe的表达模式与相关代谢酶基因的表达模式差异较大。
[Abstract]:In this study, the components and contents of organic acids in fruit development of jujube were determined by high performance liquid chromatography-diode array detector (HPLC-DAD). At the same time, spectrophotometric analysis was used to develop a pair (malate dehydrogenase NAD-MDHN, phosphoenolpyruvate carboxylase (PEPCP), malate enzyme (NADP-MEE)). The activity of citrate synthase (CSN) and other metabolic enzymes were determined. The relative expression of major organic acid-related regulatory genes in jujube fruit was detected and analyzed by qRT-PCR. A HPLC method for the determination of organic acids in jujube fruits was established. The contents of 7 organic acids and VC standard curve were all above 0.998 9. The relative standard deviation of the precision was 0.98. The relative standard deviation of the monitor was 0.88 and 2.19%. The recoveries were between 91.35% and 105.74%. The results showed that the order of organic acid content in mature jujube fruit was malic acid citrate tartrate oxalic acid fumaric acid and the highest content of malic acid was 19.21 mg 路g ~ (-1). Accounting for 89.76% of the total acid content in the same period; In the process of fruit development, except tartaric acid. The contents of malic acid and citric acid increased first and then decreased. (2) malate dehydrogenase NAD-MDH) and citric acid synthase (CSS). The change of activity was basically consistent with that of malic acid and citric acid. All showed the trend of rising with fruit development and decreasing with fruit maturation. The activity of malate enzyme NADP-MEE was reversed, and the correlation analysis showed that the change of malate content was positively correlated with the activity of NAD-MDH. There was a negative correlation between NADP-ME activity and malic acid content. The activity of CS was positively correlated with the content of citric acid, and the correlation coefficient was 0.9260; However, the activity of phosphoenolpyruvate carboxylase (PEPC) was not correlated with malic acid and citric acid content. The activities of NADP-ME and CS played an important role in the metabolism of organic acids in jujube fruits, but PEPC had little effect on the development of jujube fruits. The relative expression level of citric acid synthase gene (ZjCSA) was similar to that of citric acid content, which increased first and then decreased. The accumulation of citric acid was mainly regulated by the ZjCS gene and the malate dehydrogenase gene ZJ Mdh. The expression patterns of phosphoenolpyruvate carboxylase gene (ZjPEPC) and malic enzyme gene (ZjMe) were different in jujube and jujube fruit. The accumulation of malic acid was mainly catalyzed by NAD-MDH and regulated by ZjMdh gene, while the malic acid metabolized ZjMdh in jujube fruit. The expression patterns of ZjPEPC and ZjMe were different from those of related metabolic enzymes.
【学位授予单位】：塔里木大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S665.1

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