本文關(guān)鍵詞：脂氧合酶在薄皮甜瓜香氣物質(zhì)合成中的作用　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 脂氧合酶 薄皮甜瓜 香氣 茉莉酸甲酯處理 瞬時表達(dá)

【摘要】：薄皮甜瓜(Cucumis melo var.makuwa Makino)因其果實香氣馥郁、口感甜脆而深受消費者喜歡。香氣作為薄皮甜瓜的重要品質(zhì)特征,直接影響消費者的選擇。甜瓜果實香氣組成復(fù)雜,其中酯類物質(zhì)是果實香氣的重要組成成分,其合成途徑主要包括氨基酸途徑和脂氧合酶(lipoxygenase,LOXs)途徑。氨基酸途徑是支鏈酯類合成的主要途徑,LOX途徑是部分植物中合成直鏈香氣物質(zhì)的主要途徑,但LOX在薄皮甜瓜中直鏈酯類合成過程中起多大作用尚不明確。本文以香氣濃郁的薄皮甜瓜"玉美人"(YMR)、香氣較淡的薄皮甜瓜"翠寶"(CB)、香氣較淡的薄皮甜瓜變種梢瓜(SHAO)(C.melo var.conomon)、香氣較淡的的薄皮甜瓜變種菜瓜(CAI)(C.meloL.var.flexuosusNaud)為試材,研究了 LOX及其基因家族成員在甜瓜果實直鏈香氣物質(zhì)合成中的作用;同時,利用實時熒光定量技術(shù),研究了采后外源施用茉莉酸甲酯(MeJA)對LOXs基因家族成員的表達(dá)調(diào)控。對LOXs基因家族成員CmLOX03和CmLOX04進(jìn)行克隆及生物信息學(xué)分析,通過Gateway技術(shù)構(gòu)建CmLOX03和CmLOX04的超表達(dá)載體,利用根癌農(nóng)桿菌介導(dǎo)在薄皮甜瓜果實中進(jìn)行瞬時表達(dá),研究了 CmLOX003和CmLOX04在甜瓜果實直鏈香氣物質(zhì)合成中的作用。主要研究結(jié)果如下:1.利用氣相色譜-質(zhì)譜聯(lián)用儀(GC-MS)測定4個類型甜瓜果實的香氣成分,共檢測鑒定出87種揮發(fā)性香氣物質(zhì)。"玉美人"的總香氣成分含量和總酯類物質(zhì)含量最高。梢瓜和菜瓜則分別含有較高的揮發(fā)性醇、醛類物質(zhì)。主成分分析表明,"玉美人"、"翠寶"、梢瓜和菜瓜由主成分1區(qū)分為兩個類型。乙酸乙酯、乙酸苯甲酯、薄荷醇和反,順-2,6-壬二烯醛是幾種主要影響4種類型甜瓜香氣品質(zhì)的揮發(fā)性香氣物質(zhì)。梢瓜和菜瓜的醛類物質(zhì)含量較高,但是LOX酶活性較低;"玉美人"果實中LOX酶活性顯著高于其他3個類型甜瓜。這些結(jié)果表明,薄皮甜瓜中LOX酶活性和醛類物質(zhì)的積累呈負(fù)相關(guān),和酯類物質(zhì)的含量呈正相關(guān)。2.通過實時熒光定量PCR技術(shù)(qPCR)對18個CmLOXs基因在不同芳香類型的薄皮甜瓜中相對表達(dá)量進(jìn)行分析。酯類含量較高的"玉美人"中的CmLOX05、CmLOX10、CmLOX11和CmLOX16表達(dá)量要顯著高于其他品種;CmLOX08、CmLOX09和CmLOX17則在"翠寶"中具有較高的表達(dá)量;醇類含量較高梢瓜中CmLOX02、CmLOX03、CmLOX04 CmLOX12、CmLOX15和CmLOX1 的表達(dá)量較高;CmLOX07、CmLOX13和CmLOX14則在菜瓜中表達(dá)量顯著高于其他品種。3.以芳香濃郁的薄皮甜瓜"玉美人"為試材,通過添加LOX的前體和抑制劑進(jìn)行果肉圓片孵育試驗,研究哪些CmLOXs成員在薄皮甜瓜直鏈酯類合成中起作用。結(jié)果表明,添加LOX前體亞油酸(LA)和亞麻酸(LeA)處理誘導(dǎo)了果肉中揮發(fā)性醛類物質(zhì)和直鏈酯類物質(zhì)的積累。LOX酶活性受LA和LeA誘導(dǎo)顯著升高且被兩種LOX抑制劑顯著抑制,與直鏈酯類物質(zhì)含量積累呈正相關(guān)。CmLOX03、CmXOX03、CmLOX04、CmLOX11、CmLOX12、CmLOX16 和 CmLOX18 等 8 個 CmLOX 基因家族成員受LOX前體誘導(dǎo)而表達(dá)量顯著上調(diào)且不同程度受到LOX抑制劑的抑制而下調(diào),這些基因可能和薄皮甜瓜直鏈香氣物質(zhì)合成相關(guān)。由于CmLOX04、CmLOX05、CmLOX11和CmLOX15成熟期果肉中表達(dá)量較低,這表明其在香氣合成中作用有限。因此推測,CmLOX03、CmLOX12、CmLOX16和CmLOX18可能是甜瓜果實直鏈香氣物質(zhì)合成過程中起主要作用的基因,可以作為與薄皮甜瓜酯類香氣物質(zhì)合成相關(guān)的候選基因來進(jìn)行研究。4.以"玉美人"甜瓜為試材,通過采后外源施用MeJA,研究了 MeJA處理對LOX基因家族成員的表達(dá)調(diào)控。結(jié)果表明,采后外源施用MeJA促進(jìn)果實中LOX酶活性升高。未經(jīng)MeJA處理和MeJA處理的甜瓜中CmLOX03和CmLOX18表達(dá)量隨著時間增長呈先升高后下降趨勢。MeJA處理上調(diào)了 CmLOX03的表達(dá)并且使其表達(dá)高峰提前。CmLOX18轉(zhuǎn)錄水平受MeJA誘導(dǎo)而顯著升高,CmLOX18表達(dá)量在MeJA處理后第1天達(dá)到最高。CmLOX09和CmLOX16表達(dá)量在貯藏期間下調(diào);MeJA處理進(jìn)一步抑制CmLOX09的表達(dá),但是顯著上調(diào)了 CmLOX16的表達(dá)量。5.選擇與香氣合成相關(guān)的CmLOX03和與其序列相似性最高的CmLOX04作為候選基因進(jìn)行克隆和生物信息學(xué)分析。CmLOX03和CmLOX04蛋白都是親水性蛋白,分子量大小分別為99.634 kDa和99.860 kDa,等電點分別為6.18和5.99。CmLOX03的氨基酸序列與甜瓜基因組數(shù)據(jù)庫中的GLOX03的氨基酸序列之間相似性為85.97%,與厚皮甜瓜LOX1(DQ267934.2)的氨基酸序列之間有31個氨基酸的不同(相似性96.47%),與厚皮甜瓜LOX2(GQ386815.1)的氨基酸序列之間有4個氨基酸的不同(相似性99.54%);而CmLOX04的編碼區(qū)氨基酸序列與甜瓜基因組數(shù)據(jù)庫中的GLOX04氨基酸序列完全相同。CmLOX003和CmLOX04蛋白均含有PLAT_LH2和LOX保守結(jié)構(gòu)域,預(yù)測兩者的亞細(xì)胞定位在細(xì)胞質(zhì)中。6.利用Gateway技術(shù)構(gòu)建了CmLOX0 和CmLOX04的超表達(dá)載體,通過根癌農(nóng)桿菌介導(dǎo)的方法在薄皮甜瓜果實中進(jìn)行瞬時表達(dá)。導(dǎo)入超表達(dá)載體p-CmLOX03的甜瓜果實中CmLOX03表達(dá)量顯著升高;同時,兩種主要的直鏈酯類乙酸乙酯和乙酸己酯含量升高;p-CmLOX04的導(dǎo)入使得CmLOX04的表達(dá)量顯著升高但并不影響直鏈酯類物質(zhì)的積累。注射超表達(dá)載體p-CmLOX03和p-CmLOX04的甜瓜果肉組織LOX酶活性與對照相比差異并不顯著。因此,初步確定了 CmLOX03可能是參與薄皮甜瓜中直鏈酯類合成的重要基因,而CmLOX04則不參與直鏈酯類的合成。
[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.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S652

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