李果实苹果酸转运体的克隆表达及其有机酸的关联性分析

发布时间：2017-12-28 00:29

本文关键词：李果实苹果酸转运体的克隆表达及其有机酸的关联性分析　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

【摘要】：有机酸是决定果实品质的重要因子,参与了果实风味、色泽、香气的形成,在果实中的组成、转运与积累机理是果实品质研究的重要课题。果实细胞中,苹果酸转运蛋白tDT(tonoplast dicarboxylate transporter)和苹果酸跨膜离子通道蛋白ALMT(almuminum-activated malate transporter)基因家族介导胞质中苹果酸跨膜转运,对果实有机酸积累与降解机理研究具有重要意义。本研究通过'皇冠李'、'西瓜李'、'黑琥珀'和'葡萄李'等四个代表性品种,应用超高效液相色谱(UPLC)进行果实有机酸组分与含量的分析;通过同源克隆了一个液泡膜苹果酸转运蛋白tDT-like基因和两个苹果酸转运离子通道ALMT4-like、ALMT9-like基因,并应用实时荧光定量PCR技术检测tDT-like、AMM74-like和ALMT9-like基因在4个代表性品种中的5个发育阶段的基因表达水平,并通过pearson相关分析和回归建模系统地分析了李果实细胞苹果酸跨膜转运基因与各有机酸组分的关联性,主要研究结果如下:1.李果实生长发育过程中pH变化呈V型变化,TA呈倒V型变化。黑琥珀、皇冠李、西瓜李的pH变化V型趋势明显,葡萄李变化较小。pH值大小顺序为:成熟期,葡萄李黑琥珀≈西瓜李皇冠李,完熟期,葡萄李黑琥珀皇冠李≈西瓜李;TA变化与pH相似,黑琥珀、皇冠李、西瓜李的T倒V趋势明显,葡萄李变化较小,成熟期和完熟期果实的总酸大小顺序为:西瓜李皇冠李黑琥珀葡萄李。高酸和低酸品种的差异主要表现为低酸品种酸降较快或时间较长,反之则酸降较慢或时间较短。2.苹果酸是李果实中主要的有机酸,占总酸含量的57%～84%,李属于典型的苹果酸型果实;其次是酒石酸,个别品种(如西瓜李)高达18%～36%;而果实中的柠檬酸、琥珀酸、草酸、乙酸、富马酸只以少量或微量存在。4个测试品成熟期果实苹果酸含量顺序为:皇冠李西瓜李≈黑琥珀葡萄李。3.苹果酸转运体基因对不同品种果实苹果酸的调控作用有所差异。tDT-like基因对高苹果酸含量品种(如皇冠李、西瓜李)果实酸的积累起到一定程度的正向调控作用,对低苹果酸含量品种(如葡萄李)的调控作用不明显。ALMT9-like基因对不同品种李果实苹果酸的积累均表现为负向调控作用。ALMT4-like基因对皇冠李、西瓜李、葡萄李的苹果酸积累起到起促进作用,但对黑琥珀的苹果酸积累起到负向调控作用。4.苹果酸转运体与有机酸组分的关联性分析结果显示,ALMT4-like基因与苹果酸、琥珀酸显著相关,ALMT9-like基因与苹果酸、柠檬酸显著相关,与草酸极显著相关。表明果实细胞中ALMT4-like和ALMT9-like基因除转运苹果酸外,可能还参与琥珀酸、柠檬酸等组分的转运。
[Abstract]:Organic acid is an important factor determining fruit quality. It is involved in the formation of fruit flavor, color and aroma. The mechanism of composition, transportation and accumulation in fruit is an important topic of fruit quality research. Fruit cell, malate transporter tDT (tonoplast dicarboxylate transporter) and malic acid transmembrane ion channel protein ALMT (almuminum-activated malate transporter) gene mediated by cytoplasmic malate transport across the membrane, has important significance to research on fruit organic acid accumulation and degradation of machine. The study by Lee 'crown' and 'Lee' and 'watermelon Black Amber' and 'Lee' four grape cultivars, using ultra performance liquid chromatography (UPLC) for analysis of organic acids in fruit and content; by homologous cloning of a vacuolar malate transporter gene and tDT-like two malate transporter ALMT4-like ion channel, ALMT9-like gene, 5 stages of development and application of real time fluorescent quantitative PCR detection of tDT-like, AMM74-like and ALMT9-like genes in 4 representative varieties of gene expression level, and through Pearson correlation analysis and regression modeling and systematic analysis of the plum fruit cell malic acid Association cross membrane transporter genes and different organic acids, the main results are as follows: a V type change in the process of growth and development of pH 1. changes of plum fruit, TA presents an inverted V curve. The trend of pH change of Black Amber, crown plum and watermelon plum is obvious, and the change of grape plum is smaller. The size of the pH value of the order: the mature period, Li Li is watermelon Grape Black Amber crown plum, ripe stage, Li Li is Grape Black Amber crown watermelon Lee; the change of TA and pH is similar to that of Black Amber plum, plum, crown watermelon T inverted V trend is obvious, grape Li small changes, the total acid sequence of the mature stage and ripe stage of fruit is watermelon: Li Li Li Grape Black Amber crown. The difference of high acid and low acid variety mainly showed that the acid of low acid decreased faster or longer, and on the contrary, the acid decreased slowly or the time was shorter. 2. malic acid was the main organic acid of plum fruit, the total acid content of 57% ~ 84%, Li belongs to the typical type of fruit malic acid; followed by tartaric acid, individual species (such as watermelon Lee) up to 18% ~ 36%; and in the fruit of citric acid, succinic acid, oxalic acid, acetic acid, fumaric acid in small or trace amounts. 4 test product maturity fruit malic acid content in the order: crown Li Li Li is Black Amber grape watermelon. 3. the regulation of malic acid transporter gene on malic acid in different varieties was different. TDT-like gene played a positive regulatory role in the accumulation of acid in fruits of high malic acid content (such as crown plum, watermelon and plum), and the regulation effect on low malic acid varieties (such as grape and plum) was not obvious. The ALMT9-like gene showed negative regulation on the accumulation of malic acid in different varieties of plum fruit. ALMT4-like gene played a role in promoting malic acid accumulation in crown plum, watermelon plum and grape plum, but played a negative role in malic acid accumulation in Black Amber. 4. the correlation analysis between malic acid transporter and organic acid components showed that ALMT4-like gene was significantly correlated with malic acid and succinic acid. ALMT9-like gene was significantly correlated with malic acid and citric acid, and highly correlated with oxalate. It is suggested that the ALMT4-like and ALMT9-like genes in the fruit cells may also be involved in the transport of succinic and citric acid components, except for the transport of malic acid.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S662.3

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