洋水仙香气成分的鉴定与香气形成机制研究

发布时间：2018-03-19 05:00

本文选题：洋水仙　切入点：香气成分　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

【摘要】：洋水仙(Narissus spp.)花大色艳,花型多变,叶姿潇洒飘逸,是世界著名的观赏花卉。洋水仙栽培品种众多,但是多数品种花香较淡或者无香,品种间花香差异较大。为阐明洋水仙不同品种间花香存在差异的原因及其香气形成机制,以浓香型洋水仙'新娘皇冠'和淡香型洋水仙'胡德山'为材料,鉴定了两种洋水仙的香气成分,确定了洋水仙致香的关键成分——乙酸苯甲酯,通过分析乙酸苯甲酯合成关键酶(醇酰基转移酶)、乙酸苯甲酯合成前体(苯甲醇)与乙酸苯甲酯之间的关系,结合醇酰基转移酶基因的克隆及表达分析,从生理生化及分子生物学水平,综合探讨了不同品种洋水仙花香存在差异的原因,初步研究了洋水仙香气形成的机制,为洋水仙花香育种研究提供了理论参考。主要研究结果如下:1.利用顶空固相微萃取(HS-SPME)和气相色谱-质谱联用(GC-MS)技术,分析了洋水仙花开放过程中香气成分及含量的变化,结果表明:浓香型品种'新娘皇冠'主要的香气物质类别是萜烯类、酯类和醇类,主要的香气成分是(z)-β-罗勒烯、乙酸苯甲酯、苯甲酸甲酯、别罗勒烯、桉叶油醇,特征香气成分中贡献最大的是乙酸苯甲酯和(z)-β-罗勒烯。淡香型品种'胡德山'主要的香气物质类别是萜烯类和醇类,主要的香气成分是(z)-β-罗勒烯、别罗勒烯、β-月桂烯、对苯二甲醚、桉叶油醇,特征香气成分中贡献最大的是(z)-β-罗勒烯。浓香型与淡香型洋水仙香气的差异主要是酯类化合物种类及含量的差异,酯类化合物对洋水仙香气形成有着重要的影响,乙酸苯甲酯是洋水仙致香的关键成分,决定洋水仙花香的浓淡以及是否具有甜香的特征。2.利用DTNB比色法研究了洋水仙花开放过程中醇酰基转移酶(AAT)活性的变化,两种洋水仙AAT酶活性随着花的开放呈现先升后降的趋势,从花蕾期到盛花期,酶活性逐渐升高,到衰败期又有所下降。浓香品种'新娘皇冠'花开放过程中酯类化合物的含量与AAT酶活性呈现正相关,淡香品种'胡德山'酯类物质的含量与AAT酶活性之间并无明显的相关性。3.通过乙酸乙酯溶剂萃取法结合气相色谱-质谱联用技术,分析了洋水仙花开放过程中内源苯甲醇含量的变化。浓香品种'新娘皇冠'花开放的4个花期均萃取到苯甲醇,其含量与乙酸苯甲酯的含量呈现正相关,而淡香品种'胡德山' 4个花期均没有萃取到苯甲醇,苯甲醇的缺失是'胡德山'不能合成乙酸苯甲酯的原因。乙酸苯甲酯的合成受苯甲醇和AAT酶活性的共同影响,苯甲醇供应不足或AAT酶活性较低均会使乙酸苯甲酯的合成受阻。在苯甲醇底物供应充足的基础上,同时拥有较高的AAT酶活性,是大量合成乙酸苯甲酯的关键。4.通过外源苯甲醇处理淡香品种'胡德山'花开放过程中不同花期的花朵,研究了苯甲醇处理之后'胡德山'合成乙酸苯甲酯的情况。结果表明:在外源苯甲醇充足的情况下,'胡德山' 4个花期均萃取到一定含量的乙酸苯甲酯,说明淡香型洋水仙也具有合成乙酸苯甲酯的能力。研究解决淡香型洋水仙自身苯甲醇底物供应不足的办法,是使淡香型洋水仙具有浓郁甜香的关键。5.利用RT-PCR与RACE技术,从'新娘皇冠'和'胡德山'花中分别克隆得到一条醇酰基转移酶(AAT)基因,分别命名为NtAAT-BC和NtAAT-MD。两基因间氨基酸同源性达到98%,同属中保守性较高,与其他科属物种的AAT氨基酸同源性相对偏低,NtAAT-BC和NtAAT-MD均含有不同物种间AAT基因普遍存在且高度保守的HXXXD和FGWG基序,可以初步确定已成功从两种洋水仙中分离出AAT基因。6.采用实时荧光定量PCR技术分析了两种洋水仙花开放过程中醇酰基转移酶(AAT)基因的表达模式,结果表明:AAT基因在洋水仙不同品种和花开放的不同时期存在着表达差异。'新娘皇冠' AAT基因的表达模式与AAT酶活性、酯类化合物的变化趋势一致,'新娘皇冠' AAT基因很可能参与了 '新娘皇冠'酯类化合物的合成调控。AAT基因的差异表达可能影响洋水仙不同品种间酯类物质的合成。
[Abstract]:Narcissus (Narissus spp.) large showy flowers, flowers and Ye Zi, chic and elegant, is the world famous ornamental flower Narcissus cultivation. Many varieties, but most varieties of flowers is light or no incense, flowers larger among varieties. Differences between different varieties of Narcissus in order to clarify the differences in the formation mechanism of floral deposit and its reasons aroma to Luzhou Narcissus' bride crown 'and pink Narcissus' Hood' as material, two kinds of aroma components of Narcissus were identified and determined by the key components of Narcissus fragrance - benzyl acetate, through the analysis of acetic acid benzene ester synthesis key enzyme (AAT), benzyl acetate the synthesis of precursor (benzyl alcohol) and benzyl acetate, combined with the cloning and expression analysis of alcohol acyltransferase gene, from the physiology and biochemistry and molecular biology level, a comprehensive discussion of the different varieties of ocean water Xian differences in flowers A preliminary study on the causes, mechanism of Narcissus aroma formation, provides a theoretical reference for narcissus flower breeding. The main results are as follows: 1. using headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) technology, analyzes the changes of aroma components and contents of Narcissus in the process of opening up show: Luzhou 'crown' main varieties bride aroma categories are terpenes, esters and alcohols, the main aroma components of the (z) - beta ocimene, benzyl acetate, methyl benzoate, alloocimene, eucalyptus oil alcohol, with characteristic aroma components was the largest of benzyl acetate and (z) - beta ocimene. Pink variety 'Hood' main aroma categories are terpenes and alcohols, the main aroma components of the (z) - beta ocimene, alloocimene, myrcene, two of benzene ether, eudesmol, characteristic aroma components the contribution of the The (z) - beta ocimene. Differences in Luzhou and pink Narcissus aroma is mainly different kinds and contents of ester compounds, ester compounds have important influence on the formation of Narcissus aroma, benzyl acetate is a key component of Narcissus by incense, Narcissus flowers shade and decision whether.2. has the characteristics of sweet use DTNB Narcissus in the opening process of alcohol acyltransferase Colorimetry (AAT) activity changes of two kinds of Narcissus AAT activity with flower decreased, from bud stage to flowering, enzyme activity gradually increased, and declined to a period of decline at present positive correlation content and AAT activity of Luzhou varieties' crown 'bride flowers esters in the process between the content and AAT activity of fragrant varieties' Hood' esters of.3. have no obvious correlation with ethyl acetate Chromatography-mass spectrometry method of solvent extraction combined with gas, analyzes the changes of endogenous Benzenemethanol Narcissus in the opening process. 4 flowering fragrant varieties' bride 'crown of flower opening were extracted by benzyl alcohol, are positively related to the content and content of benzyl acetate, and fragrant varieties' Hood' 4 flowering were not extracted by benzyl alcohol, benzyl alcohol is lack of reason 'Hood' can't synthesis of benzyl acetate. The synthesis of benzyl acetate together by benzyl alcohol and AAT enzyme activity, benzyl alcohol shortage or AAT low enzyme activity can cause the synthesis of acetic acid benzylester blocked in benzene methanol substrate supply adequate basis, the activity of AAT and have high, different flowers key.4. synthesis of phenyl acetate methyl benzene methanol treated by exogenous fragrant varieties' Hood 'flowers in the process of benzene Situation of Mount Hood 'synthesis of phenyl acetate methyl alcohol treatment after'. The results showed that: in the exogenous benzyl alcohol is sufficient, the 'Hood' 4 stage was extracted into ethyl methyl benzene content, indicating that pink Narcissus is synthesis of benzyl acetate. To study and solve the pink Narcissus itself Benzenemethanol substrate supply problems, is the key to.5. pink Narcissus with rich sweet using RT-PCR and RACE technology, from the 'bride crown' and 'Hood' flowers were cloned from an alcohol acyltransferase (AAT) gene, named NtAAT-BC and NtAAT-MD. amino acid homology between the two loci reached 98%. High conservative belong to, and AAT amino acid species homology is relatively low, NtAAT-BC and NtAAT-MD contained AAT gene among species are ubiquitous and highly conserved motifs HXXXD and FGWG, can be the beginning of Step two has been successfully determined from Narcissus isolated AAT.6. gene by real-time fluorescence quantitative PCR analysis of two kinds of Narcissus in the opening process of alcohol acyltransferase (AAT) gene expression patterns, the results show that the different stages of AAT gene in Narcissus varieties and flower open exists expression pattern and activity of AAT differential expression of AAT gene. The bride 'crown, the same trend of ester compounds, may affect the ocean water Xian among different varieties of esters synthesis synthesis regulation of.AAT gene expression between' bride 'AAT' crown gene is likely the bride 'crown esters to participate.

【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】：S682.21

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