本文關(guān)鍵詞： 棗 有機酸 酶活性 基因表達　出處：《塔里木大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究以駿棗和酸棗為試材,采用高效液相色譜-二極管陣列檢測器法(HPLC-DAD)對棗果實發(fā)育過程中有機酸組分與含量進行了檢測,同時利用分光光度發(fā)對(蘋果酸脫氫酶(NAD-MDH)、磷酸烯醇式丙酮酸羧化酶(PEPC)、蘋果酸酶(NADP-ME)、檸檬酸合成酶(CS))等代謝酶活性進行了檢測,以及通過qRT-PCR對棗果實發(fā)育過程中的主要有機酸相關(guān)調(diào)控基因的相對表達量進行了動態(tài)檢測分析。結(jié)果表明:(1)建立了棗果實中有機酸含量的HPLC測定方法,7種有機酸組分和Vc標(biāo)準(zhǔn)曲線的(R2)均在0.998 9以上;精密度檢測出其相對標(biāo)準(zhǔn)偏差為0.98%~4.77%(n=5);重復(fù)性檢測結(jié)果顯示器相對標(biāo)準(zhǔn)偏差值為0.88%~2.19%(n=5);回收率均在91.35%~105.74%之間;得出在成熟期棗果實中的有機酸含量由高到低順序依次為:蘋果酸檸檬酸酒石酸奎寧酸草酸富馬酸,其中蘋果酸含量最高可達19.21 mg·g-1,占同期總酸含量的89.76%;在果實發(fā)育過程中,除酒石酸外,蘋果酸和檸檬酸含量的變化均呈先升高后降低的趨勢。(2)蘋果酸脫氫酶(NAD-MDH)和檸檬酸合成酶(CS)活性變化與蘋果酸和檸檬酸含量的變化趨勢基本一致,均呈現(xiàn)伴隨果實發(fā)育而上升,伴隨果實成熟而降低的趨勢,蘋果酸酶(NADP-ME)活性變化則相反。相關(guān)性分析表明:NAD-MDH活性變化與蘋果酸含量變化呈正相關(guān),NADP-ME活性變化與蘋果酸含量變化呈負相關(guān);CS活性變化與檸檬酸含量達到極顯著正相關(guān),相關(guān)系數(shù)可達0.9260;而磷酸烯醇式丙酮酸羧化酶(PEPC)活性變化與蘋果酸和檸檬酸含量相關(guān)性不大。本研究認為:NAD-MDH、NADP-ME和CS活性對棗果實中有機酸代謝發(fā)揮重要作用,而PEPC作用不大。(3)在棗果實發(fā)育過程中,檸檬酸合成酶基因(ZjCS)的相對相對表達量的變化趨勢與檸檬酸含量的變化趨勢基本一致,呈先上升后降低的趨勢,檸檬酸的積累主要受ZjCS基因的調(diào)控作用。而蘋果酸脫氫酶基因(ZjMdh)、磷酸烯醇式丙酮酸羧化酶基因(ZjPEPC)和蘋果酸酶基因(ZjMe)的表達模式在酸棗和駿棗中則表現(xiàn)不一,在酸棗果實中,蘋果酸的積累主要受到NAD-MDH酶催化同時受到ZjMdh基因的調(diào)控作用。而在駿棗果實中蘋果酸代謝的ZjMdh、ZjPEPC和ZjMe的表達模式與相關(guān)代謝酶基因的表達模式差異較大。
[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.
【學(xué)位授予單位】：塔里木大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S665.1

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