脂氧合酶在薄皮甜瓜香气物质合成中的作用

发布时间：2018-01-16 09:07

  本文关键词：脂氧合酶在薄皮甜瓜香气物质合成中的作用　出处：《沈阳农业大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 脂氧合酶 薄皮甜瓜 香气 茉莉酸甲酯处理 瞬时表达

【摘要】：薄皮甜瓜(Cucumis melo var.makuwa Makino)因其果实香气馥郁、口感甜脆而深受消费者喜欢。香气作为薄皮甜瓜的重要品质特征,直接影响消费者的选择。甜瓜果实香气组成复杂,其中酯类物质是果实香气的重要组成成分,其合成途径主要包括氨基酸途径和脂氧合酶(lipoxygenase,LOXs)途径。氨基酸途径是支链酯类合成的主要途径,LOX途径是部分植物中合成直链香气物质的主要途径,但LOX在薄皮甜瓜中直链酯类合成过程中起多大作用尚不明确。本文以香气浓郁的薄皮甜瓜"玉美人"(YMR)、香气较淡的薄皮甜瓜"翠宝"(CB)、香气较淡的薄皮甜瓜变种梢瓜(SHAO)(C.melo var.conomon)、香气较淡的的薄皮甜瓜变种菜瓜(CAI)(C.meloL.var.flexuosusNaud)为试材,研究了 LOX及其基因家族成员在甜瓜果实直链香气物质合成中的作用;同时,利用实时荧光定量技术,研究了采后外源施用茉莉酸甲酯(MeJA)对LOXs基因家族成员的表达调控。对LOXs基因家族成员CmLOX03和CmLOX04进行克隆及生物信息学分析,通过Gateway技术构建CmLOX03和CmLOX04的超表达载体,利用根癌农杆菌介导在薄皮甜瓜果实中进行瞬时表达,研究了 CmLOX003和CmLOX04在甜瓜果实直链香气物质合成中的作用。主要研究结果如下:1.利用气相色谱-质谱联用仪(GC-MS)测定4个类型甜瓜果实的香气成分,共检测鉴定出87种挥发性香气物质。"玉美人"的总香气成分含量和总酯类物质含量最高。梢瓜和菜瓜则分别含有较高的挥发性醇、醛类物质。主成分分析表明,"玉美人"、"翠宝"、梢瓜和菜瓜由主成分1区分为两个类型。乙酸乙酯、乙酸苯甲酯、薄荷醇和反,顺-2,6-壬二烯醛是几种主要影响4种类型甜瓜香气品质的挥发性香气物质。梢瓜和菜瓜的醛类物质含量较高,但是LOX酶活性较低;"玉美人"果实中LOX酶活性显著高于其他3个类型甜瓜。这些结果表明,薄皮甜瓜中LOX酶活性和醛类物质的积累呈负相关,和酯类物质的含量呈正相关。2.通过实时荧光定量PCR技术(qPCR)对18个CmLOXs基因在不同芳香类型的薄皮甜瓜中相对表达量进行分析。酯类含量较高的"玉美人"中的CmLOX05、CmLOX10、CmLOX11和CmLOX16表达量要显著高于其他品种;CmLOX08、CmLOX09和CmLOX17则在"翠宝"中具有较高的表达量;醇类含量较高梢瓜中CmLOX02、CmLOX03、CmLOX04 CmLOX12、CmLOX15和CmLOX1 的表达量较高;CmLOX07、CmLOX13和CmLOX14则在菜瓜中表达量显著高于其他品种。3.以芳香浓郁的薄皮甜瓜"玉美人"为试材,通过添加LOX的前体和抑制剂进行果肉圆片孵育试验,研究哪些CmLOXs成员在薄皮甜瓜直链酯类合成中起作用。结果表明,添加LOX前体亚油酸(LA)和亚麻酸(LeA)处理诱导了果肉中挥发性醛类物质和直链酯类物质的积累。LOX酶活性受LA和LeA诱导显著升高且被两种LOX抑制剂显著抑制,与直链酯类物质含量积累呈正相关。CmLOX03、CmXOX03、CmLOX04、CmLOX11、CmLOX12、CmLOX16 和 CmLOX18 等 8 个 CmLOX 基因家族成员受LOX前体诱导而表达量显著上调且不同程度受到LOX抑制剂的抑制而下调,这些基因可能和薄皮甜瓜直链香气物质合成相关。由于CmLOX04、CmLOX05、CmLOX11和CmLOX15成熟期果肉中表达量较低,这表明其在香气合成中作用有限。因此推测,CmLOX03、CmLOX12、CmLOX16和CmLOX18可能是甜瓜果实直链香气物质合成过程中起主要作用的基因,可以作为与薄皮甜瓜酯类香气物质合成相关的候选基因来进行研究。4.以"玉美人"甜瓜为试材,通过采后外源施用MeJA,研究了 MeJA处理对LOX基因家族成员的表达调控。结果表明,采后外源施用MeJA促进果实中LOX酶活性升高。未经MeJA处理和MeJA处理的甜瓜中CmLOX03和CmLOX18表达量随着时间增长呈先升高后下降趋势。MeJA处理上调了 CmLOX03的表达并且使其表达高峰提前。CmLOX18转录水平受MeJA诱导而显著升高,CmLOX18表达量在MeJA处理后第1天达到最高。CmLOX09和CmLOX16表达量在贮藏期间下调;MeJA处理进一步抑制CmLOX09的表达,但是显著上调了 CmLOX16的表达量。5.选择与香气合成相关的CmLOX03和与其序列相似性最高的CmLOX04作为候选基因进行克隆和生物信息学分析。CmLOX03和CmLOX04蛋白都是亲水性蛋白,分子量大小分别为99.634 kDa和99.860 kDa,等电点分别为6.18和5.99。CmLOX03的氨基酸序列与甜瓜基因组数据库中的GLOX03的氨基酸序列之间相似性为85.97%,与厚皮甜瓜LOX1(DQ267934.2)的氨基酸序列之间有31个氨基酸的不同(相似性96.47%),与厚皮甜瓜LOX2(GQ386815.1)的氨基酸序列之间有4个氨基酸的不同(相似性99.54%);而CmLOX04的编码区氨基酸序列与甜瓜基因组数据库中的GLOX04氨基酸序列完全相同。CmLOX003和CmLOX04蛋白均含有PLAT_LH2和LOX保守结构域,预测两者的亚细胞定位在细胞质中。6.利用Gateway技术构建了CmLOX0 和CmLOX04的超表达载体,通过根癌农杆菌介导的方法在薄皮甜瓜果实中进行瞬时表达。导入超表达载体p-CmLOX03的甜瓜果实中CmLOX03表达量显著升高;同时,两种主要的直链酯类乙酸乙酯和乙酸己酯含量升高;p-CmLOX04的导入使得CmLOX04的表达量显著升高但并不影响直链酯类物质的积累。注射超表达载体p-CmLOX03和p-CmLOX04的甜瓜果肉组织LOX酶活性与对照相比差异并不显著。因此,初步确定了 CmLOX03可能是参与薄皮甜瓜中直链酯类合成的重要基因,而CmLOX04则不参与直链酯类的合成。
[Abstract]:Muskmelon (Cucumis melo var.makuwa Makino) because of its fruit fragrance, crisp and sweet taste by consumers love. As an important feature of muskmelon aroma quality, directly affect consumer choice. Muskmelon fruit aroma composition is complex, the esters are important components of fruit aroma, the synthesis ways mainly include the way and lipoxygenase amino acid synthase (lipoxygenase, LOXs). Amino acid pathway is the main pathway for the synthesis of branched chain esters, LOX pathway is the main way to part of the plant to synthesize straight chain compounds, but LOX in Muskmelon amylose ester synthesis process effect is not clear. The aroma of Muskmelon "Yumeiren" (YMR), a light aroma of Muskmelon "cuibao" (CB), a light aroma of Muskmelon Varieties of melon shoot (SHAO) (C.melo var.conomon), the lighter the aroma of Muskmelon Varieties of vegetables Melon (CAI) (C.meloL.var.flexuosusNaud) as test materials, studied the effect of LOX gene family members and in Muskmelon Fruit Aroma in the synthesis of amylose; at the same time, using real-time fluorescence quantitative techniques to study the Postharvest application of Exogenous Methyl Jasmonate (MeJA) expression of LOXs gene family members. Cloning and analysis bioinformatics of LOXs gene family members CmLOX03 and CmLOX04, over expression vector based on Gateway technology CmLOX03 and CmLOX04, using Agrobacterium mediated transient expression in muskmelon fruit, the effects of CmLOX003 and CmLOX04 in Muskmelon Fruit straight chain aroma synthesis. The main results are as follows: 1. by gas chromatography-mass spectrometry (GC-MS) determination of aroma components of 4 types of muskmelon fruit were detected, identified 87 volatile aroma substances. The total content of aroma components of Yumeiren "and the total The highest content of esters. Volatile alcohol, melon and squash shoots respectively contain higher aldehydes. Principal component analysis showed that the "jade beauty", "Cui Bao, Shao Gua and melons by principal component 1 is divided into two types. Ethyl acetate, benzyl acetate, menthol and anti clockwise, -2,6- nonadienal aldehydes are some of the main influence the aroma quality of 4 types of muskmelon aroma. The content of aldehydes and Caigua Shao Gua is high, but LOX low enzyme activity; LOX enzyme activity of Yumeiren fruit was significantly higher than the other 3 types of muskmelon. These results indicated that LOX was negatively related to enzyme activity and aldehydes thin the accumulation of muskmelon, and was positively related to.2. content of esters by real-time fluorescence quantitative PCR (qPCR) of 18 CmLOXs genes in different types of fragrant muskmelon were analyzed. The relative expression of esters with high content of" jade beauty "in CmLOX0 5, CmLOX10, CmLOX11 and CmLOX16 expression was significantly higher than that of other varieties; CmLOX08, CmLOX09 and CmLOX17 level was higher in the "cuibao"; higher alcohol content in shoots of melon CmLOX02, CmLOX03, CmLOX04, CmLOX12, CmLOX15 and the high expression of CmLOX1; CmLOX07, CmLOX13 and CmLOX14 expression in Caigua in the amount of.3. was significantly higher than that of other varieties with fragrant Muskmelon "jade beauty" as the test material, by fruitdiscs incubation experiment by precursor and addition of inhibitor of LOX, on which CmLOXs members in Muskmelon amylose ester synthesis plays an important role. The results show that the addition of LOX precursor linoleic acid (LA) and flax acid (LeA) accumulation of.LOX activity induced volatile aldehydes in the pulp and straight chain esters by LA and induced by LeA was significantly increased and the two kinds of LOX inhibitors significantly inhibited, and straight chain esters matter content accumulation was positively related to.C MLOX03, CmXOX03, CmLOX04, CmLOX11, CmLOX12, CmLOX16 and CmLOX18 8 CmLOX gene family members LOX precursor induced expression was up-regulated and not affected by the inhibitor of LOX and down-regulation of these genes, and muskmelon amylose aroma synthesis. Because of CmLOX04, CmLOX05, CmLOX11 and CmLOX15 expression of mature during the period of pulp was low, indicating that in the aroma synthesis limited effect. So that, CmLOX03, CmLOX12, CmLOX16 and CmLOX18 might play an important role in Muskmelon Fruit Aroma in the process of synthesis of straight chain genes, and can be used as muskmelon aroma esters synthesis of candidate genes related to.4. was studied by jade beauty "muskmelon as test material, through the Postharvest exogenous MeJA, MeJA on the regulation of the expression of LOX gene family. The results showed that exogenous MeJA promote fruit after harvest Increase in LOX activity. Without MeJA treatment and MeJA treatment in Muskmelon CmLOX03 and CmLOX18 expression increase with time was increased first and then decreased.MeJA up-regulated the expression of CmLOX03 and its expression peak early transcription level of.CmLOX18 induced by MeJA significantly increased, the expression of CmLOX18 MeJA in first days after treatment and reached the highest.CmLOX09 and CmLOX16 expression decreased during storage; the expression of MeJA further inhibition of CmLOX09, but significantly up-regulated the expression of CmLOX16.5. and aroma synthesis related CmLOX03 and its sequence similarity to the high CmLOX04 as a candidate gene cloning and bioinformatics analysis of.CmLOX03 and CmLOX04 protein is a hydrophilic protein molecule. The size was 99.634 kDa and 99.860 kDa, isoelectric point were the amino acid sequence of the muskmelon genome database of 6.18 and 5.99.CmLOX03 in Between the GLOX03 amino acid sequence similarity of 85.97%, and muskmelon LOX1 (DQ267934.2) 31 amino acids between different amino acid sequences (96.47% similarity), and Muskmelon (GQ386815.1 LOX2) 4 amino acids between different amino acid sequences (99.54% similarity); and the amino acid sequence of GLOX04 encoding region the amino acid sequence of muskmelon genome database in CmLOX04 is identical to.CmLOX003 and CmLOX04 protein contained PLAT_LH2 and LOX conserved domain, subcellular localization prediction both in the cytoplasm of.6. using Gateway technology to construct the expression vector of CmLOX0 and CmLOX04, through agrobacterium mediated method in Muskmelon Fruit instantaneous expression. Overexpression of CmLOX03 expression vector into p-CmLOX03 in muskmelon fruit was significantly higher; at the same time, two kinds of straight chain esters of ethyl acetate and acetic acid hexyl ester content Increased; p-CmLOX04 import makes the expression of CmLOX04 increased significantly but does not affect the accumulation of straight chain esters. Over expression of LOX injection muskmelon flesh tissue enzyme activity vector p-CmLOX03 and p-CmLOX04 compared with the control group the difference was not significant. Therefore, initially identified CmLOX03 may be an important gene involved in Muskmelon amylose ester synthesis, synthesis CmLOX04 is not involved in straight chain esters.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S652

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