不同工艺铁观音香气形成的生化及分子生物学机制研究
|
摘要
铁观音是乌龙茶中的极品，其以香气独特驰名中外，关于铁观音香气形成的化学机制研究尚多，而其酶学、分子生物学机制的研究鲜有报道。本研究以铁观音为供试材料，研究了铁观音香气形成的生化及分子生物学机制研究。通过对不同工艺各加工工序铁观音香气总量、组分、成分的测定与分析，探讨不同工艺铁观音香气总量、组分、成分的动态变化；通过对不同工艺各加工工序铁观音β-glucosidase活性测定与分析，探讨不同工艺铁观音β-glucosidase活性的动态变化及其与香气总量、组分、成分间的相关性；克隆了铁观音茶树香气相关基因cβ-glucosidase并对其相关的生物信息进行预测和分析；开展不同工艺各加工工序铁观音β-glucosidase基因的定量表达与分析。综合分析铁观音香气变化及其与β-glucosidase活性、基因表达间的相关性，揭示铁观音香气形成的机理。研究结果如下：
1.不同工艺铁观音香气的动态变化
探讨不同工艺铁观音香气的动态变化。结果表明，(1)晒青阶段：低温轻发酵做青工艺中“轻晒青”阶段，铁观音主要赋香成分均有所增加，其中有效促进了α-法尼烯、β-紫罗酮、香叶基丙酮、茉莉酮、雪松烯等主要香气成分大量形成；传统自然做青工艺中“重晒青”同样促进铁观音主要赋香成分形成，其增加幅度不及“轻晒青”。(2)摇青阶段：低温轻发酵做青工艺中“轻摇青”阶段，铁观音主要赋香成分均有所增加，其中有效促进了橙花叔醇、α-法尼烯、β-紫罗酮、香叶基丙酮、茉莉酮、丁酸反-2-己烯酯、苯甲酸叶醇酯大量形成；传统自然做青工艺中“重摇青”在促进铁观音主要赋香成分形成的同时，有效促进了橙花叔醇、α-法尼烯、β-法尼烯、雪松烯、L-薄荷醇、β-紫罗酮、香叶基丙酮、丁酸反-2-己烯酯、苯甲酸叶醇酯、乙酸苯乙酯、己酸己酯大量形成；传统自然做青工艺中“重摇青”铁观音香气峰面积总值为低温轻发酵做青工艺中“轻摇青”的1.39倍，则“重摇青”较“轻摇青”更有效地促进铁观音香气的形成。(3)摊青阶段：低温轻发酵做青工艺中“长时薄摊青”的整个过程，有效促进了β-榄香烯、茉莉酮、丁酸反-2-己烯酯、乙酸苯乙酯、2-丙基-1-庚醇大量形成，摊青24h以后，香气总量虽达到最高值，但主要特征香气成分如橙花叔醇、α-法尼烯含量有所减少；传统自然做青工艺中“短时厚摊青”发酵过程中各主要特征香气成分含量与摇青结束时基本保持同一水平。
2.不同季节、不同风格铁观音成品茶香气差异
探讨不同季节、不同风格铁观音成品茶香气差异。结果表明，(1)不同季节铁观音香气差异：秋季加工铁观音在各香气组分含量上均高于春季加工铁观音，该季节加工铁观音成品茶的橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、乙酸苯乙酯、茉莉酸甲酯、己酸己酯、吲哚、香叶基丙酮、β-紫罗酮、茉莉酮、二十烷等特殊香气成分含量显著高于春季加工铁观音成品茶。(2)不同风格铁观音香气差异：清香正韵型、青韵型、显酸型成品茶及传统浓香型铁观音茶坯香气组分均以醇类、烯类化合物为主，其共有的重要香气成分为橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、乙酸苯乙酯、β-紫罗酮且含量较高；传统浓香型铁观音主要香气成分橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、己酸己酯、吲哚、香叶基丙酮、苯甲酸叶醇酯、二十烷含量高于清香型铁观音；清香正韵型铁观音主要香气成分α-法尼烯、二十烷、β-法尼烯含量高于其他风格铁观音，α-法尼烯高于清香青韵、显酸型；清香青韵型顺罗勒烯含量较为突出；清香显酸型乙酸苯乙酯、茉莉酮、3,7-甲基-1,5,7-三辛烯-3-醇、雪松烯含量较突出。
3.不同风格铁观音成品茶香气成分与感官审评的关系
分析了铁观音成品茶香气成分与感官审评的关系。结果表明，传统浓香型铁观音橙花叔醇、α-法尼烯2种重要特征香气含量最高，其他多种香气成分含量均高于清香型铁观音，由此其香气浓度、强度、持久性表现优异，而清爽度、纯度较差；清香正韵型铁观音橙花叔醇、α-法尼烯2种重要特征香气含量高于清香青韵、显酸型，而丁酸反-2-己烯酯、乙酸苯乙酯、吲哚、茉莉酸甲酯、己酸己酯、β-紫罗酮、顺-罗勒烯等多种香气含量低于其他风格铁观音，由此其香气纯度、清爽度表现优异；清香青韵型香气成分含量居中为多，由此其香气表现不突出；清香显酸型香气总量最高，但其橙花叔醇、α-法尼烯2种重要特征香气含量较低，而其他多种香气成分含量均高于其他风格铁观音，由此其香气浓度虽好，但纯度、清爽度、强度均较差，略有闷杂特征。
研究得出铁观音主要赋香成分为橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、乙酸苯乙酯、吲哚、茉莉酸甲酯、己酸己酯、香叶基丙酮、苯甲酸叶醇酯、β-紫罗酮、顺-罗勒烯、二十烷、茉莉酮、β-法尼烯、3,7-甲基-1,5,7-三辛烯-3-醇、雪松烯等。而铁观音香气给予品茗者“如花带果”感受的化学依据在于：似兰花香，如茉莉酸甲酯；似桂花香，如顺-罗勒烯及多种单萜类和倍半萜类物质；似栀子花香，如芳樟醇、香叶醇、苯甲酸甲酯；似梅花香如乙酸苯甲酯；似蜜桃香，如内酯。已测得铁观音香气成分或与其相同，或经异构、转化能形成该类香气，由此作为铁观音形成浓郁花果香气的化学依据。
4.不同季节、不同工艺、不同叶位铁观音β-glucosidase酶活性变化差异
讨探了不同季节、不同工艺、不同叶位铁观音β-glucosidase酶活性变化差异。结果表明，不同季节铁观音β-glucosidase酶活性变化差异如下：低温轻发酵做青工艺下，春季铁观音成熟叶β-glucosidase酶活性可达秋季铁观音的1.4-2.6倍，其变化辐度大于秋季，而两季铁观音成熟叶β-glucosidase酶活性变化趋势略有不同，摊青期间两者酶活性表现为“此起彼伏”变化特点；传统自然做青工艺下，春季铁观音β-glucosidase酶活性可达秋季铁观音的1.7-2.2倍，且春季酶活性变化辐度大于秋季，而两季铁观音成熟叶β-glucosidase酶活性的试样变化趋势一致。由此可知，春季铁观音成熟叶β-glucosidase酶活性远高于秋季铁观音，其变辐大于秋季。
不同工艺铁观音β-glucosidase酶活性变化差异如下：低温轻发酵工艺下铁观音β-glucosidase酶活性高于传统自然做青工艺；春季不同工艺铁观音β-glucosidase酶活性变化趋势较一致，即晒青阶段有所上升，摇青阶段呈“降-升-降-(升)”的波形变化趋势，摊青至4-5h酶活性下降而后上升；秋季不同工艺铁观音β-glucosidase酶活性变化趋势有此消彼长的变化特点，且传统自然做青工艺变辐大于低温轻发酵做青工艺。
不同叶位铁观音β-glucosidase酶活性变化差异如下：春季不同叶位铁观音β-glucosidase酶活性高低差异不明显，而秋季不同叶位铁观音β-glucosidase酶活性高低表现为成熟叶>老叶>嫩叶；春秋两季、轻重做青的铁观音不同叶位β-glucosidase酶活性多数在摇青前中期达到最高值；春秋两季铁观音嫩叶位β-glucosidase酶活性在不同工艺下酶活性变辐均较大。
5.铁观音cβ-glucosidase克隆
从铁观音中克隆出cβ-glucosidase片段，该cDNA序列长度1477bp，包含开放阅读框(ORF)共有1284个碱基组成，编码428个氨基酸。在3’RACE与保守区重叠碱基数为267bp，非编码区3’RACE有186bp，与其他植物同源60%-80%。已将该基因序列登录GenBank，登录号为HQ679938.2。
6.铁观音cβ-glucosidase生物信息学分析
分析了铁观音cβ-glucosidase相关生物信息。结果表明，铁观音cβ-glucosidase蛋白属于碱性、稳定、亲水蛋白。铁观音cβ-glucosidase定位细胞质，存在跨膜结构域，属于跨膜蛋白，但由于没有信号肽即为非分泌蛋白。另外，其磷酸化位点有20个，其中在C末端有Ser磷酸化位点，预测Ser磷酸化可能与铁观音cβ-glucosidase蛋白构象变化、核定位调节、细胞周期之间存在的密切联系。
7.不同工艺铁观音cβ-glucosidase的表达量差异
探讨了不同工艺铁观音cβ-glucosidase的表达量差异，结果显示：在低温轻发酵、传统自然做青工艺中，晒青及摇青阶段cβ-glucosidase相对表达量逐步增加，晒青期间该基因的相对表达量与鲜叶比较差异不显著，摇青结束其变化达到极显著水平；而摊青前期表达量变化不显著，至摊青时间达6-10h，cβ-glucosidase相对表达量均达到最高值，与鲜叶、晒青叶、摇青叶的相对表达量比较，差异均达到极显著水平。综上说明铁观音加工工艺促进了cβ-glucosidase的表达，摊青至一定程度该基因表达信号最强，表达量最高，随着摊青的延续，基因表达减弱。
8.不同工艺铁观音香气变化、β-glucosidase酶活性、基因定量表达的相关性
探讨了不同工艺铁观音香气变化、β-glucosidase酶活性、基因定量表达的相关性。结果表明，不同工艺铁观音β-glucosidase酶活性与香气变化关系表现为：β-glucosidase酶活性与香气含量成指数负相关。低温轻发酵做青工艺及传统自然工艺中，铁观音成熟叶β-glucosidase酶活性与香气总量、醇系香气含量、特征香气橙花叔醇含量的关系表现为：晒青阶段酶活性的提高伴随着香气总量、醇系香气含量、特征香气橙花叔醇含量的提高；摇青前期酶活性大幅上升而伴随着游离态芳香物质的大量生成；摊青阶段，酶活性的变化趋势与香气的变化趋势基本一致。
不同工艺铁观音cβ-glucosidase定量表达与酶活性关系表现为：在低温轻发酵做青工艺、传统自然做青工艺中，晒青阶段cβ-glucosidase相对表达量有所增加，酶活性也显著上升；摇青结束相对表达量持续增加，而酶活性显著下降；摊青阶段，铁观音基因相对表达量与酶活性邻间变化率正负值相同，则两者的变化趋势一致。
不同工艺铁观音cβ-glucosidase定量表达与香气变化关系表现为：在低温轻发酵及传统自然做青工艺的晒青及摇青阶段，cβ-glucosidase相对表达量与香气峰面积总值、醇系香气峰面积值、橙花叔醇峰面积值均保持不断上升的趋势；摊青过程，基因相对表达量与香气峰面积总值变化趋势保持一致，而摊青后期基因表达量的显著变化没有引起醇系香气成分的相应变化，而是略呈此消彼长的变化趋势。
Tieguanyin is highest grade of oolong tea, and its unique aroma is known at home andabroad. About studies on the chemical mechanism of tieguanyin aroma formed are many, but itsenzymology, molecular biology mechanism research reported rarely.In this study were used astieguanyin materials to study biochemistry and molecular mechanism of tieguanyin aromaformation.It determined and analysised tieguanyin’s aroma substances, components, the totalamount in different processing phase,and explored the dynamic change of tieguanyin’s aromasubstances, components, the total amount.Tieguanyin’s β-glucosidase activity in differentprocessing phase was determed and analysised,and it explored the dynamic change oftieguanyin’s β-glucosidase activity and the correlations among β-glucosidase and aromasubstances, components,the total amount.Tea aroma gene of cβ-glucosidase was cloned fromTieguanyin and its biological information was forecasted and analysised. The quantitativeexpression of Tieguanyin’s cβ-glucosidase in different processing phase was analyzed.On thebasis of the studies above, the correlations of Tieguanyin tea aroma change, β-glucosidaseactivity, cβ-glucosidase expression were analysised comprehensively,and aroma formation’smechanism was revealed.The main results shows as follows:
1. The dynamic change of tieguanyin aroma in different processes
The dynamic change of tieguanyin aroma in different process was explored in this study.The results shows that:(1) The sun-withering stage: In the process of tea leaves-makingtechnology of the degree of light fermentation in low temperature,tieguanyin main aromacompositions increased in sun-withering lightly processing, and the sun-withering lightlyprocessing promoted effectively main aroma substances to form in quantity,such as alphaFarnesene,beta Ionone,geranylacetone, Jasmone,cedrene. In the process of traditional tealeaves-making technology in the natural conditions,the sun-withering heavily processing alsopromoted tieguanyin main aroma to form, and the increase rate was inferior to the sun-witheringlightly.(2) The shaking tea leaves stage: In the process of tea leaves-making technology of thedegree of light fermentation in low temperature,tieguanyin main aroma compositions increasedin shaking tea leaves lightly processing, and shaking tea leaves lightly processing promotedeffectively main aroma substances to form in quantity,such as Nerolidol,alpha Farnesene,betaLonone,Geranylacetone,Jasmone,trans-2-Hexenyl butyate,cis-3-Hexenyl benzoate.In the processof traditional tea leaves-making technology in the natural conditions,the shaking tea leavesheavily processing also promoted effectively main aroma substances to form in quantity,such asNerolidol,alpha Farnesen,bate Farnesene,Cedrene,L-Menthol,beta Lonone,Geranylacetone,trans-2-Hexenyl butyate,cis-3-Hexenyl benzoate,Phenethyl acetate,Hexyl hexanoate. The Total aromaforming in shaking tea leaves heavily processing was that forming in shaking tea leaves lightlyprocessing1.39times,then the former more effectively than the latter promotes the formation oftieguanyin aroma.(3) The spreading tea leaves stage: In the process of tea leaves-makingtechnology of the degree of light fermentation in low temperature,spreading tea leaves thinly fora long time promoted effectively main aroma substances to form in quantity,such as bate Elemene, Jasmone, trans-2-Hexenyl butyate, Phenethyl acetate,2-Propyl-1-heptanol. Afterspreading tea leaves for24h, although total aroma reached a high level, but the maincharacteristics aroma such as Nerolidol aroma,alpha Farnesene content reduced down.
2. Differences of tieguanyin tea aroma in different seasons and different stylesDifferences of tieguanyin tea aroma in different seasons and different styles were explored. Theresults shows that:(1) Differences of different seasons tieguanyin aroma: the aroma contents ofautumn processing tieguanyin was higher than spring. The main characteristics aroma of autumnprocessing tieguanyin was significantly higher than spring,such as Nerolidol, alpha Farnesene,trans-2-Hexenyl butyate, Phenethyl acetate, Methyl dihydrojasmonate, Hexyl hexanoate, Indole,Geranylacetone, beta Lonone, Jasmone,N-Eicosane.(2) Differences of different style tieguanyinaroma: the fragrant and lingering charm type, the tea leaves fagrant and lingering charm type, thesour fragrant type and the traditional and highly scented type tieguanyin had two majorconstituents of alcohol and Alkenes.They had common aroma substances and highlycontents,such as Nerolidol, alpha Farnesene,trans-2-Hexenyl butyate,Phenethyl acetate,betaLonone. The main aroma substances of the traditional and highly scented type tieguanyin werehigher than the fragrant type such as Nerolido, alpha Farnesene, trans-2-Hexenyl butyate, Hexylhexanoate, Indole, Geranylacetone, cis-3-Hexenyl benzoate, Eicosane.The aroma substances ofthe fragrant and lingering charm type tieguanyin were higher than other types such as alphaFarnesene,bate Farnesene, N-Eicosane.The Ocimene content of the tea leaves fagrant andlingering charm type was more prominent.The Phenethyl acetate, Jasmone,3,7-methyl-1,5,7-three octene-3-alcohol and Cedrene content of the sour fragrant type were more prominent.
3.The relations of different Tieguanyin styles’ aroma substances and sensory evaluation
The relations of different Tieguanyin styles’ aroma substances and sensory evaluation wereexplored. The results showed that: The significant characteristics aroma Nerolidol and alphaFarnesene contents of the traditional and highly scented type were the highest.Other aromasubstances contents were higher than the fragrant type.Thus its aroma’s concentration, intensityand persistance performed excellently,but elegant degrees and purity performed poorly. Thesignificant characteristics aroma Nerolidol and alpha Farnesene contents of the traditional andhighly scented type were the highest. The relations of different Tieguanyin styles’ aromasubstances and sensory evaluation were explored. The results showed that: The significantcharacteristics aroma Nerolidol and alpha Farnesene contents of the traditional and highlyscented type were the highest.Other aroma substances contents were higher than the fragranttype.Thus its aroma’s concentration, intensity and persistance performed excellently,but freshdegrees and purity performed poorly.The significant characteristics aroma Nerolidol and alphaFarnesene contents of the fragrant and lingering charm type were higher than other the fragranttypes.But it had many aroma substances’ contents were lower than other types such astrans-2-Hexenyl butyate,Phenethyl acetate,Indole,Methyl dihydrojasmonate,Hexyl hexanoate,beta Lonone,Ocimene.So its fresh degrees and purity performed excellently. The aromasubstances’ contents of the tea leaves fagrant and lingering charm type got moderate level.Thetotal aroma contects of the sour fragrant type was highest,but the significant characteristicsaroma Nerolidol and alpha Farnesene contents were lower and many others aroma substancescontects were higher than other types.So its concentration was better but purity and fresh degreesand intensity were bader,then it has miscellaneous characteristics.
The Research obtained the main aroma substances of tieguanyin were Nerolidol,alphaFarnesene,trans-2-Hexenyl butyate,Phenethyl acetate,Indole,Methyl dihydrojasmonate,Hexylhexanoate,Geranylacetone,cis-3-Hexenyl benzoate,betaLonone,Ocimene,N-Eicosane,Jasmone,Bate Farnesene,3,7-methyl-1,5,7-three octene-3-alcohol,Cedrene and so on. The chemical basisthat Tieguanyin aroma giving ardent tea people feeling of " fragrance of flower and fruit aroma " lied in: Orchid aroma contains Methyl dihydrojasmonate. Osmanthus aroma contains Ocimene,Monoterpenoids and Sesquiterpenoids. Gardenia aroma contains Linalool, Geraniol and Methylbenzoate..Plum aroma contains Benzyl acetate.Honey peach aroma contains lactone.Thetieguanyin aroma substances determined were the identical with them, or proceed heterogeneousand transformation to form them.
4. Differences of tieguanyin β-glucosidase enzyme activity changed in differentseasons,different processing and different places’ leaves
Differences of tieguanyin β-glucosidase enzyme activity changed in differentseasons,different processing and different places’ leaves were explored. The results showed thatdifferences of tieguanyin β-glucosidase enzyme activity changed in different seasons were asfollows:In the process of the tea leaves-making technology of the degree of light fermentation inlow temperature, β-glucosidase enzyme activity of spring tieguanyin mature leaves was autumn’s1.4-2.6times and rangeability was larger than autumn’s.Then in two seasons tieguanyin matureleaves beta glucosidas enzyme activity change trend were a little different,and themperformed“as one falls another rises” change characteristics. In the process of the traditional tealeaves-making technology in the natural conditions, β-glucosidase enzyme activity of springtieguanyin mature leaves was autumn’s1.7-2.2times and rangeability was larger than autumn’s.Then in two seasons tieguanyin mature leaves beta glucosidas enzyme activity change trend wereidentical. Therefore, β-glucosidase enzyme activity of spring tieguanyin mature leaves washigher than autumn’s and rangeability was larger than autumn’s.
Differences of tieguanyin β-glucosidase enzyme activity changed in different processingwere as follows:In the process of the tea leaves-making technology of the degree of lightfermentation in low temperature, β-glucosidase enzyme activity was higher than the traditionaltea leaves-making technology in the natural conditions.In spring, β-glucosidase enzyme activitychange of different processings were identical,which ascended in the sun-withering stage,appeared the change trend”drop-rise-drop-(rise)”in the shaking tea leaves stage. In autumn,β-glucosidase enzyme activity change of different processings performed“as one falls anotherrises” change characteristics,and the rangeability of the traditional tea leaves-making technologywas larger than the tea leaves-making technology of the degree of light fermentation.
Differences of tieguanyin β-glucosidase enzyme activity change in leaves at the differentplaces were as follows:In spring, differences of β-glucosidase enzyme activity of leaves at thedifferent places were small. In autumn, differences of β-glucosidase enzyme activity of leaves atthe different places performed that mature leaves>old leaf> young leaf. β-Glucosidase enzymeactivity of leaves at the different places got the highest in former midium-term and β-glucosidaseenzyme activity of young leaves has large variation range in two seasons and technologys.
5.cDNA clonging of tieguanyin β-glucosidase
The sequence's length of tieguanyin cβ-glucosidase cloned was1477bp,including openreading frame (ORF) which contained1284base composition and encoded428amino acids. Inthe3'RACE and conservative base area overlaped for267bp, the coding areas' RACE was186bp, with other plant homologous rate was60%-80%.This genetic sequence was registered inGenBank, and the registration number was HQ679938.2.
6. Analysis of bioinformatics of tieguanyin cβ-glucosidase
Bioinformatics of tieguanyin cβ-glucosidase was analyzed. The results shows that, theprotein of tieguanyin cβ-glucosidase was alkaline, stable and belong to hydrophilic protein.Tieguanyin cβ-glucosidase located in cytoplasm and existed transmembrane domain so belong totransmembrane protein, nevertheless existed no signal peptide so wasn’t secretory protein.Inaddition, tieguanyin cβ-glucosidase had20phosphorylation sites, and Ser of phosphorylationsite lied in C-terminal. Ser of phosphorylation site was forecasted relating with tieguanyin cβ-glucosidase ‘s protein conformation change, nuclear localization regulation and cell cycle.
7. Differences of tieguanyin cβ-glucosidase expression level in different processing
Differences of tieguanyin cβ-glucosidase expression level in different processing wereexplored. The results shows that: In the process of the tea leaves-making technology of thedegree of light fermentation in low temperature and the traditional tea leaves-making technologyin the natural conditions, there had been a gradual rise in tieguanyin cβ-glucosidase expressionlevel in sun-withering and shaking tea leaves stage. No significant differences existed betweentieguanyin cβ-glucosidase expression level in sun-withering and shaking tea leaves stage andcβ-glucosidase expression level rised significantly in shaking tea leaves stage end.In earlierspreading tea leaves stage, cβ-glucosidase expression level changed significantly,and whenspreading tea leaves stund up to6-10h, cβ-glucosidase expression level reached the highest. Allthat tieguanyin tea processing technology promoted cβ-glucosidase to express,and spreading tealeaves stage to a certain extent.cβ-Glucosidase expressed level highestly,and as the continuationof spreading tea leaves, cβ-Glucosidase expression weakened.
8.The relations of different tieguanyin aroma change, β-glucosidase enzyme activity andcβ-glucosidase expression level in different processing
The relations of different tieguanyin aroma change, β-glucosidase enzyme activity andcβ-glucosidase expression level in different processing were explored. The results shows that:Tieguanyin β-glucosidase enzyme activity and aroma contents presented index negativecorrelation. In the process of the tea leaves-making technology of the degree of lightfermentation in low temperature and the traditional tea leaves-making technology in the naturalconditions,the relations of β-glucosidase enzyme activity and aroma amount, alcoholic aromacontent, the significant characteristics aroma Nerolidol content perform that:In sun-witheringstage, β-glucosidase enzyme activity improved with aroma amount, alcoholic aroma content,andthe significant characteristics aroma Nerolidol content;In former midium-term of shaking tealeaves stage, β-glucosidase enzyme activity improved with Pretreated aromatic substanceslargely;In spreading tea leaves stage, the change trend of enzyme activity and the changetendency of the aroma were identical.
The relations of β-glucosidase enzyme activity and cβ-glucosidase expression level indifferent processing perform that:In the process of the tea leaves-making technology of thedegree of light fermentation in low temperature and the traditional tea leaves-making technologyin the natural conditions, cβ-glucosidase expression level increased and β-glucosidase enzymeactivity improved significantly. In the end shaking tea leaves stage，cβ-glucosidase expressionlevel increased persistently，and β-glucosidase enzyme activity descended significantly.Inspreading tea leaves stage, the change trends of β-glucosidase enzyme activity andcβ-glucosidase expression level were identical.
The relations of cβ-glucosidase expression level and aroma contents in different processingperform that: In the sun-withering and shaking tea leaves processes of the tea leaves-makingtechnology of the degree of light fermentation in low temperature and the traditional tealeaves-making technology in the natural conditions, cβ-glucosidase expression level and aromaamount, alcoholic aroma content, the significant characteristics aroma Nerolidol content keptrising trend.In spreading tea leaves stage, the change trend of aroma amount consistented withcβ-glucosidase expression level. However, cβ-glucosidase expression level increased persistentlywhich didn’t cause alcoholic aroma content change correspondly.They appeared the changetrend”drop-rise-drop-(rise)” in the spreading tea leaves stage.
1.不同工艺铁观音香气的动态变化
探讨不同工艺铁观音香气的动态变化。结果表明，(1)晒青阶段：低温轻发酵做青工艺中“轻晒青”阶段，铁观音主要赋香成分均有所增加，其中有效促进了α-法尼烯、β-紫罗酮、香叶基丙酮、茉莉酮、雪松烯等主要香气成分大量形成；传统自然做青工艺中“重晒青”同样促进铁观音主要赋香成分形成，其增加幅度不及“轻晒青”。(2)摇青阶段：低温轻发酵做青工艺中“轻摇青”阶段，铁观音主要赋香成分均有所增加，其中有效促进了橙花叔醇、α-法尼烯、β-紫罗酮、香叶基丙酮、茉莉酮、丁酸反-2-己烯酯、苯甲酸叶醇酯大量形成；传统自然做青工艺中“重摇青”在促进铁观音主要赋香成分形成的同时，有效促进了橙花叔醇、α-法尼烯、β-法尼烯、雪松烯、L-薄荷醇、β-紫罗酮、香叶基丙酮、丁酸反-2-己烯酯、苯甲酸叶醇酯、乙酸苯乙酯、己酸己酯大量形成；传统自然做青工艺中“重摇青”铁观音香气峰面积总值为低温轻发酵做青工艺中“轻摇青”的1.39倍，则“重摇青”较“轻摇青”更有效地促进铁观音香气的形成。(3)摊青阶段：低温轻发酵做青工艺中“长时薄摊青”的整个过程，有效促进了β-榄香烯、茉莉酮、丁酸反-2-己烯酯、乙酸苯乙酯、2-丙基-1-庚醇大量形成，摊青24h以后，香气总量虽达到最高值，但主要特征香气成分如橙花叔醇、α-法尼烯含量有所减少；传统自然做青工艺中“短时厚摊青”发酵过程中各主要特征香气成分含量与摇青结束时基本保持同一水平。
2.不同季节、不同风格铁观音成品茶香气差异
探讨不同季节、不同风格铁观音成品茶香气差异。结果表明，(1)不同季节铁观音香气差异：秋季加工铁观音在各香气组分含量上均高于春季加工铁观音，该季节加工铁观音成品茶的橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、乙酸苯乙酯、茉莉酸甲酯、己酸己酯、吲哚、香叶基丙酮、β-紫罗酮、茉莉酮、二十烷等特殊香气成分含量显著高于春季加工铁观音成品茶。(2)不同风格铁观音香气差异：清香正韵型、青韵型、显酸型成品茶及传统浓香型铁观音茶坯香气组分均以醇类、烯类化合物为主，其共有的重要香气成分为橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、乙酸苯乙酯、β-紫罗酮且含量较高；传统浓香型铁观音主要香气成分橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、己酸己酯、吲哚、香叶基丙酮、苯甲酸叶醇酯、二十烷含量高于清香型铁观音；清香正韵型铁观音主要香气成分α-法尼烯、二十烷、β-法尼烯含量高于其他风格铁观音，α-法尼烯高于清香青韵、显酸型；清香青韵型顺罗勒烯含量较为突出；清香显酸型乙酸苯乙酯、茉莉酮、3,7-甲基-1,5,7-三辛烯-3-醇、雪松烯含量较突出。
3.不同风格铁观音成品茶香气成分与感官审评的关系
分析了铁观音成品茶香气成分与感官审评的关系。结果表明，传统浓香型铁观音橙花叔醇、α-法尼烯2种重要特征香气含量最高，其他多种香气成分含量均高于清香型铁观音，由此其香气浓度、强度、持久性表现优异，而清爽度、纯度较差；清香正韵型铁观音橙花叔醇、α-法尼烯2种重要特征香气含量高于清香青韵、显酸型，而丁酸反-2-己烯酯、乙酸苯乙酯、吲哚、茉莉酸甲酯、己酸己酯、β-紫罗酮、顺-罗勒烯等多种香气含量低于其他风格铁观音，由此其香气纯度、清爽度表现优异；清香青韵型香气成分含量居中为多，由此其香气表现不突出；清香显酸型香气总量最高，但其橙花叔醇、α-法尼烯2种重要特征香气含量较低，而其他多种香气成分含量均高于其他风格铁观音，由此其香气浓度虽好，但纯度、清爽度、强度均较差，略有闷杂特征。
研究得出铁观音主要赋香成分为橙花叔醇、α-法尼烯、丁酸反-2-己烯酯、乙酸苯乙酯、吲哚、茉莉酸甲酯、己酸己酯、香叶基丙酮、苯甲酸叶醇酯、β-紫罗酮、顺-罗勒烯、二十烷、茉莉酮、β-法尼烯、3,7-甲基-1,5,7-三辛烯-3-醇、雪松烯等。而铁观音香气给予品茗者“如花带果”感受的化学依据在于：似兰花香，如茉莉酸甲酯；似桂花香，如顺-罗勒烯及多种单萜类和倍半萜类物质；似栀子花香，如芳樟醇、香叶醇、苯甲酸甲酯；似梅花香如乙酸苯甲酯；似蜜桃香，如内酯。已测得铁观音香气成分或与其相同，或经异构、转化能形成该类香气，由此作为铁观音形成浓郁花果香气的化学依据。
4.不同季节、不同工艺、不同叶位铁观音β-glucosidase酶活性变化差异
讨探了不同季节、不同工艺、不同叶位铁观音β-glucosidase酶活性变化差异。结果表明，不同季节铁观音β-glucosidase酶活性变化差异如下：低温轻发酵做青工艺下，春季铁观音成熟叶β-glucosidase酶活性可达秋季铁观音的1.4-2.6倍，其变化辐度大于秋季，而两季铁观音成熟叶β-glucosidase酶活性变化趋势略有不同，摊青期间两者酶活性表现为“此起彼伏”变化特点；传统自然做青工艺下，春季铁观音β-glucosidase酶活性可达秋季铁观音的1.7-2.2倍，且春季酶活性变化辐度大于秋季，而两季铁观音成熟叶β-glucosidase酶活性的试样变化趋势一致。由此可知，春季铁观音成熟叶β-glucosidase酶活性远高于秋季铁观音，其变辐大于秋季。
不同工艺铁观音β-glucosidase酶活性变化差异如下：低温轻发酵工艺下铁观音β-glucosidase酶活性高于传统自然做青工艺；春季不同工艺铁观音β-glucosidase酶活性变化趋势较一致，即晒青阶段有所上升，摇青阶段呈“降-升-降-(升)”的波形变化趋势，摊青至4-5h酶活性下降而后上升；秋季不同工艺铁观音β-glucosidase酶活性变化趋势有此消彼长的变化特点，且传统自然做青工艺变辐大于低温轻发酵做青工艺。
不同叶位铁观音β-glucosidase酶活性变化差异如下：春季不同叶位铁观音β-glucosidase酶活性高低差异不明显，而秋季不同叶位铁观音β-glucosidase酶活性高低表现为成熟叶>老叶>嫩叶；春秋两季、轻重做青的铁观音不同叶位β-glucosidase酶活性多数在摇青前中期达到最高值；春秋两季铁观音嫩叶位β-glucosidase酶活性在不同工艺下酶活性变辐均较大。
5.铁观音cβ-glucosidase克隆
从铁观音中克隆出cβ-glucosidase片段，该cDNA序列长度1477bp，包含开放阅读框(ORF)共有1284个碱基组成，编码428个氨基酸。在3’RACE与保守区重叠碱基数为267bp，非编码区3’RACE有186bp，与其他植物同源60%-80%。已将该基因序列登录GenBank，登录号为HQ679938.2。
6.铁观音cβ-glucosidase生物信息学分析
分析了铁观音cβ-glucosidase相关生物信息。结果表明，铁观音cβ-glucosidase蛋白属于碱性、稳定、亲水蛋白。铁观音cβ-glucosidase定位细胞质，存在跨膜结构域，属于跨膜蛋白，但由于没有信号肽即为非分泌蛋白。另外，其磷酸化位点有20个，其中在C末端有Ser磷酸化位点，预测Ser磷酸化可能与铁观音cβ-glucosidase蛋白构象变化、核定位调节、细胞周期之间存在的密切联系。
7.不同工艺铁观音cβ-glucosidase的表达量差异
探讨了不同工艺铁观音cβ-glucosidase的表达量差异，结果显示：在低温轻发酵、传统自然做青工艺中，晒青及摇青阶段cβ-glucosidase相对表达量逐步增加，晒青期间该基因的相对表达量与鲜叶比较差异不显著，摇青结束其变化达到极显著水平；而摊青前期表达量变化不显著，至摊青时间达6-10h，cβ-glucosidase相对表达量均达到最高值，与鲜叶、晒青叶、摇青叶的相对表达量比较，差异均达到极显著水平。综上说明铁观音加工工艺促进了cβ-glucosidase的表达，摊青至一定程度该基因表达信号最强，表达量最高，随着摊青的延续，基因表达减弱。
8.不同工艺铁观音香气变化、β-glucosidase酶活性、基因定量表达的相关性
探讨了不同工艺铁观音香气变化、β-glucosidase酶活性、基因定量表达的相关性。结果表明，不同工艺铁观音β-glucosidase酶活性与香气变化关系表现为：β-glucosidase酶活性与香气含量成指数负相关。低温轻发酵做青工艺及传统自然工艺中，铁观音成熟叶β-glucosidase酶活性与香气总量、醇系香气含量、特征香气橙花叔醇含量的关系表现为：晒青阶段酶活性的提高伴随着香气总量、醇系香气含量、特征香气橙花叔醇含量的提高；摇青前期酶活性大幅上升而伴随着游离态芳香物质的大量生成；摊青阶段，酶活性的变化趋势与香气的变化趋势基本一致。
不同工艺铁观音cβ-glucosidase定量表达与酶活性关系表现为：在低温轻发酵做青工艺、传统自然做青工艺中，晒青阶段cβ-glucosidase相对表达量有所增加，酶活性也显著上升；摇青结束相对表达量持续增加，而酶活性显著下降；摊青阶段，铁观音基因相对表达量与酶活性邻间变化率正负值相同，则两者的变化趋势一致。
不同工艺铁观音cβ-glucosidase定量表达与香气变化关系表现为：在低温轻发酵及传统自然做青工艺的晒青及摇青阶段，cβ-glucosidase相对表达量与香气峰面积总值、醇系香气峰面积值、橙花叔醇峰面积值均保持不断上升的趋势；摊青过程，基因相对表达量与香气峰面积总值变化趋势保持一致，而摊青后期基因表达量的显著变化没有引起醇系香气成分的相应变化，而是略呈此消彼长的变化趋势。
Tieguanyin is highest grade of oolong tea, and its unique aroma is known at home andabroad. About studies on the chemical mechanism of tieguanyin aroma formed are many, but itsenzymology, molecular biology mechanism research reported rarely.In this study were used astieguanyin materials to study biochemistry and molecular mechanism of tieguanyin aromaformation.It determined and analysised tieguanyin’s aroma substances, components, the totalamount in different processing phase,and explored the dynamic change of tieguanyin’s aromasubstances, components, the total amount.Tieguanyin’s β-glucosidase activity in differentprocessing phase was determed and analysised,and it explored the dynamic change oftieguanyin’s β-glucosidase activity and the correlations among β-glucosidase and aromasubstances, components,the total amount.Tea aroma gene of cβ-glucosidase was cloned fromTieguanyin and its biological information was forecasted and analysised. The quantitativeexpression of Tieguanyin’s cβ-glucosidase in different processing phase was analyzed.On thebasis of the studies above, the correlations of Tieguanyin tea aroma change, β-glucosidaseactivity, cβ-glucosidase expression were analysised comprehensively,and aroma formation’smechanism was revealed.The main results shows as follows:
1. The dynamic change of tieguanyin aroma in different processes
The dynamic change of tieguanyin aroma in different process was explored in this study.The results shows that:(1) The sun-withering stage: In the process of tea leaves-makingtechnology of the degree of light fermentation in low temperature,tieguanyin main aromacompositions increased in sun-withering lightly processing, and the sun-withering lightlyprocessing promoted effectively main aroma substances to form in quantity,such as alphaFarnesene,beta Ionone,geranylacetone, Jasmone,cedrene. In the process of traditional tealeaves-making technology in the natural conditions,the sun-withering heavily processing alsopromoted tieguanyin main aroma to form, and the increase rate was inferior to the sun-witheringlightly.(2) The shaking tea leaves stage: In the process of tea leaves-making technology of thedegree of light fermentation in low temperature,tieguanyin main aroma compositions increasedin shaking tea leaves lightly processing, and shaking tea leaves lightly processing promotedeffectively main aroma substances to form in quantity,such as Nerolidol,alpha Farnesene,betaLonone,Geranylacetone,Jasmone,trans-2-Hexenyl butyate,cis-3-Hexenyl benzoate.In the processof traditional tea leaves-making technology in the natural conditions,the shaking tea leavesheavily processing also promoted effectively main aroma substances to form in quantity,such asNerolidol,alpha Farnesen,bate Farnesene,Cedrene,L-Menthol,beta Lonone,Geranylacetone,trans-2-Hexenyl butyate,cis-3-Hexenyl benzoate,Phenethyl acetate,Hexyl hexanoate. The Total aromaforming in shaking tea leaves heavily processing was that forming in shaking tea leaves lightlyprocessing1.39times,then the former more effectively than the latter promotes the formation oftieguanyin aroma.(3) The spreading tea leaves stage: In the process of tea leaves-makingtechnology of the degree of light fermentation in low temperature,spreading tea leaves thinly fora long time promoted effectively main aroma substances to form in quantity,such as bate Elemene, Jasmone, trans-2-Hexenyl butyate, Phenethyl acetate,2-Propyl-1-heptanol. Afterspreading tea leaves for24h, although total aroma reached a high level, but the maincharacteristics aroma such as Nerolidol aroma,alpha Farnesene content reduced down.
2. Differences of tieguanyin tea aroma in different seasons and different stylesDifferences of tieguanyin tea aroma in different seasons and different styles were explored. Theresults shows that:(1) Differences of different seasons tieguanyin aroma: the aroma contents ofautumn processing tieguanyin was higher than spring. The main characteristics aroma of autumnprocessing tieguanyin was significantly higher than spring,such as Nerolidol, alpha Farnesene,trans-2-Hexenyl butyate, Phenethyl acetate, Methyl dihydrojasmonate, Hexyl hexanoate, Indole,Geranylacetone, beta Lonone, Jasmone,N-Eicosane.(2) Differences of different style tieguanyinaroma: the fragrant and lingering charm type, the tea leaves fagrant and lingering charm type, thesour fragrant type and the traditional and highly scented type tieguanyin had two majorconstituents of alcohol and Alkenes.They had common aroma substances and highlycontents,such as Nerolidol, alpha Farnesene,trans-2-Hexenyl butyate,Phenethyl acetate,betaLonone. The main aroma substances of the traditional and highly scented type tieguanyin werehigher than the fragrant type such as Nerolido, alpha Farnesene, trans-2-Hexenyl butyate, Hexylhexanoate, Indole, Geranylacetone, cis-3-Hexenyl benzoate, Eicosane.The aroma substances ofthe fragrant and lingering charm type tieguanyin were higher than other types such as alphaFarnesene,bate Farnesene, N-Eicosane.The Ocimene content of the tea leaves fagrant andlingering charm type was more prominent.The Phenethyl acetate, Jasmone,3,7-methyl-1,5,7-three octene-3-alcohol and Cedrene content of the sour fragrant type were more prominent.
3.The relations of different Tieguanyin styles’ aroma substances and sensory evaluation
The relations of different Tieguanyin styles’ aroma substances and sensory evaluation wereexplored. The results showed that: The significant characteristics aroma Nerolidol and alphaFarnesene contents of the traditional and highly scented type were the highest.Other aromasubstances contents were higher than the fragrant type.Thus its aroma’s concentration, intensityand persistance performed excellently,but elegant degrees and purity performed poorly. Thesignificant characteristics aroma Nerolidol and alpha Farnesene contents of the traditional andhighly scented type were the highest. The relations of different Tieguanyin styles’ aromasubstances and sensory evaluation were explored. The results showed that: The significantcharacteristics aroma Nerolidol and alpha Farnesene contents of the traditional and highlyscented type were the highest.Other aroma substances contents were higher than the fragranttype.Thus its aroma’s concentration, intensity and persistance performed excellently,but freshdegrees and purity performed poorly.The significant characteristics aroma Nerolidol and alphaFarnesene contents of the fragrant and lingering charm type were higher than other the fragranttypes.But it had many aroma substances’ contents were lower than other types such astrans-2-Hexenyl butyate,Phenethyl acetate,Indole,Methyl dihydrojasmonate,Hexyl hexanoate,beta Lonone,Ocimene.So its fresh degrees and purity performed excellently. The aromasubstances’ contents of the tea leaves fagrant and lingering charm type got moderate level.Thetotal aroma contects of the sour fragrant type was highest,but the significant characteristicsaroma Nerolidol and alpha Farnesene contents were lower and many others aroma substancescontects were higher than other types.So its concentration was better but purity and fresh degreesand intensity were bader,then it has miscellaneous characteristics.
The Research obtained the main aroma substances of tieguanyin were Nerolidol,alphaFarnesene,trans-2-Hexenyl butyate,Phenethyl acetate,Indole,Methyl dihydrojasmonate,Hexylhexanoate,Geranylacetone,cis-3-Hexenyl benzoate,betaLonone,Ocimene,N-Eicosane,Jasmone,Bate Farnesene,3,7-methyl-1,5,7-three octene-3-alcohol,Cedrene and so on. The chemical basisthat Tieguanyin aroma giving ardent tea people feeling of " fragrance of flower and fruit aroma " lied in: Orchid aroma contains Methyl dihydrojasmonate. Osmanthus aroma contains Ocimene,Monoterpenoids and Sesquiterpenoids. Gardenia aroma contains Linalool, Geraniol and Methylbenzoate..Plum aroma contains Benzyl acetate.Honey peach aroma contains lactone.Thetieguanyin aroma substances determined were the identical with them, or proceed heterogeneousand transformation to form them.
4. Differences of tieguanyin β-glucosidase enzyme activity changed in differentseasons,different processing and different places’ leaves
Differences of tieguanyin β-glucosidase enzyme activity changed in differentseasons,different processing and different places’ leaves were explored. The results showed thatdifferences of tieguanyin β-glucosidase enzyme activity changed in different seasons were asfollows:In the process of the tea leaves-making technology of the degree of light fermentation inlow temperature, β-glucosidase enzyme activity of spring tieguanyin mature leaves was autumn’s1.4-2.6times and rangeability was larger than autumn’s.Then in two seasons tieguanyin matureleaves beta glucosidas enzyme activity change trend were a little different,and themperformed“as one falls another rises” change characteristics. In the process of the traditional tealeaves-making technology in the natural conditions, β-glucosidase enzyme activity of springtieguanyin mature leaves was autumn’s1.7-2.2times and rangeability was larger than autumn’s.Then in two seasons tieguanyin mature leaves beta glucosidas enzyme activity change trend wereidentical. Therefore, β-glucosidase enzyme activity of spring tieguanyin mature leaves washigher than autumn’s and rangeability was larger than autumn’s.
Differences of tieguanyin β-glucosidase enzyme activity changed in different processingwere as follows:In the process of the tea leaves-making technology of the degree of lightfermentation in low temperature, β-glucosidase enzyme activity was higher than the traditionaltea leaves-making technology in the natural conditions.In spring, β-glucosidase enzyme activitychange of different processings were identical,which ascended in the sun-withering stage,appeared the change trend”drop-rise-drop-(rise)”in the shaking tea leaves stage. In autumn,β-glucosidase enzyme activity change of different processings performed“as one falls anotherrises” change characteristics,and the rangeability of the traditional tea leaves-making technologywas larger than the tea leaves-making technology of the degree of light fermentation.
Differences of tieguanyin β-glucosidase enzyme activity change in leaves at the differentplaces were as follows:In spring, differences of β-glucosidase enzyme activity of leaves at thedifferent places were small. In autumn, differences of β-glucosidase enzyme activity of leaves atthe different places performed that mature leaves>old leaf> young leaf. β-Glucosidase enzymeactivity of leaves at the different places got the highest in former midium-term and β-glucosidaseenzyme activity of young leaves has large variation range in two seasons and technologys.
5.cDNA clonging of tieguanyin β-glucosidase
The sequence's length of tieguanyin cβ-glucosidase cloned was1477bp,including openreading frame (ORF) which contained1284base composition and encoded428amino acids. Inthe3'RACE and conservative base area overlaped for267bp, the coding areas' RACE was186bp, with other plant homologous rate was60%-80%.This genetic sequence was registered inGenBank, and the registration number was HQ679938.2.
6. Analysis of bioinformatics of tieguanyin cβ-glucosidase
Bioinformatics of tieguanyin cβ-glucosidase was analyzed. The results shows that, theprotein of tieguanyin cβ-glucosidase was alkaline, stable and belong to hydrophilic protein.Tieguanyin cβ-glucosidase located in cytoplasm and existed transmembrane domain so belong totransmembrane protein, nevertheless existed no signal peptide so wasn’t secretory protein.Inaddition, tieguanyin cβ-glucosidase had20phosphorylation sites, and Ser of phosphorylationsite lied in C-terminal. Ser of phosphorylation site was forecasted relating with tieguanyin cβ-glucosidase ‘s protein conformation change, nuclear localization regulation and cell cycle.
7. Differences of tieguanyin cβ-glucosidase expression level in different processing
Differences of tieguanyin cβ-glucosidase expression level in different processing wereexplored. The results shows that: In the process of the tea leaves-making technology of thedegree of light fermentation in low temperature and the traditional tea leaves-making technologyin the natural conditions, there had been a gradual rise in tieguanyin cβ-glucosidase expressionlevel in sun-withering and shaking tea leaves stage. No significant differences existed betweentieguanyin cβ-glucosidase expression level in sun-withering and shaking tea leaves stage andcβ-glucosidase expression level rised significantly in shaking tea leaves stage end.In earlierspreading tea leaves stage, cβ-glucosidase expression level changed significantly,and whenspreading tea leaves stund up to6-10h, cβ-glucosidase expression level reached the highest. Allthat tieguanyin tea processing technology promoted cβ-glucosidase to express,and spreading tealeaves stage to a certain extent.cβ-Glucosidase expressed level highestly,and as the continuationof spreading tea leaves, cβ-Glucosidase expression weakened.
8.The relations of different tieguanyin aroma change, β-glucosidase enzyme activity andcβ-glucosidase expression level in different processing
The relations of different tieguanyin aroma change, β-glucosidase enzyme activity andcβ-glucosidase expression level in different processing were explored. The results shows that:Tieguanyin β-glucosidase enzyme activity and aroma contents presented index negativecorrelation. In the process of the tea leaves-making technology of the degree of lightfermentation in low temperature and the traditional tea leaves-making technology in the naturalconditions,the relations of β-glucosidase enzyme activity and aroma amount, alcoholic aromacontent, the significant characteristics aroma Nerolidol content perform that:In sun-witheringstage, β-glucosidase enzyme activity improved with aroma amount, alcoholic aroma content,andthe significant characteristics aroma Nerolidol content;In former midium-term of shaking tealeaves stage, β-glucosidase enzyme activity improved with Pretreated aromatic substanceslargely;In spreading tea leaves stage, the change trend of enzyme activity and the changetendency of the aroma were identical.
The relations of β-glucosidase enzyme activity and cβ-glucosidase expression level indifferent processing perform that:In the process of the tea leaves-making technology of thedegree of light fermentation in low temperature and the traditional tea leaves-making technologyin the natural conditions, cβ-glucosidase expression level increased and β-glucosidase enzymeactivity improved significantly. In the end shaking tea leaves stage，cβ-glucosidase expressionlevel increased persistently，and β-glucosidase enzyme activity descended significantly.Inspreading tea leaves stage, the change trends of β-glucosidase enzyme activity andcβ-glucosidase expression level were identical.
The relations of cβ-glucosidase expression level and aroma contents in different processingperform that: In the sun-withering and shaking tea leaves processes of the tea leaves-makingtechnology of the degree of light fermentation in low temperature and the traditional tealeaves-making technology in the natural conditions, cβ-glucosidase expression level and aromaamount, alcoholic aroma content, the significant characteristics aroma Nerolidol content keptrising trend.In spreading tea leaves stage, the change trend of aroma amount consistented withcβ-glucosidase expression level. However, cβ-glucosidase expression level increased persistentlywhich didn’t cause alcoholic aroma content change correspondly.They appeared the changetrend”drop-rise-drop-(rise)” in the spreading tea leaves stage.
引文
[1]蔡建明.安溪铁观音品质形成的生化原理初探[J].福建茶叶,1994,(1):8-11.
[2]周昱,魏宏,吴天送,等.乌龙茶“铁观音”香气成分的气相色谱/质谱分析[J].色谱,1994,12(3):355-357.
[3]于欣洋,岳文杰,李金辉,等.茶叶香气研究进展[J].茶叶科学技术,2008.(3):9-13.
[4]陈美霞,陈学森,周杰.蒸馏-萃取法与溶剂萃取法提取杏果实香气成分的比较[J].分析试验室,2005,24(3):65-70.
[5]宛晓春.茶叶生物化学[M].北京:中国农业出版社,2003.8:39-49.
[6]林正奎,华映芳,谷豫红,等.茶鲜叶挥发油化学成分的研究[J].植物学报,1982,124(2):23-28.
[7]吴勇.萜烯类化合物与茶叶香气[J].化学工程与装备,2009,(11):123-125.
[8]张长波,孙红霞,巩中军,等.植物萜类化合物的天然合成途径及其相关合酶[J].植物生理学通讯,2007,43(4):779-785.
[9]吕连梅,董尚胜.茶叶香气的研究进展[J].茶叶,2002,28(4):181-184.
[10]朱旗,施兆鹏,任春梅.绿茶香气不同提取方法的研究[J].茶叶科学,2001,21(l):38-43.
[11]孙宗保,赵杰文,邹小波,等.HS-SPME/GC-MS/GC-O对镇江香醋特征香气成分的确定[J].江苏大学学报,2010,31(2):139-144.
[12]马永昆,蒋家奎,魏永义,等.基于GC-MS与嗅闻联用的镇江香醋香气指纹图谱研究[J].食品科学,2007,28(9):496-499.
[13]夏玲君,宋焕禄.香味检测技术GC-O的应用[J].食品与发酵工业,2006,32(1):83-87.
[14]吉克温,金心怡,苏益平.乌龙茶空调制茶技术要点[J].福建茶叶,2004,(1):16.
[15]郑乃辉,姚信恩,林锦江,等.轻发酵乌龙茶工艺技术总结[J].茶叶科学技术,2004,(2):34,41.
[16]谢萍娟,李旭云.清香型铁观音的制作技术[J].中国茶叶,2006,(4):32-33.
[17]孙云,吉克温,杨江帆,等.清香型乌龙茶加工技术与配套设备[J].中国茶叶,2007,(3):9-11.
[18]孙云,吉克温,杨江帆,等.清香型乌龙茶加工技术与配套设备(续)[J].中国茶叶,2007,(4):8-10.
[19]张方舟,陈加友.清香型乌龙茶空调做青设备及加工的关键技术[J].中国茶叶,2005,(1):32-33.
[20]李宗恒,陈郁榕.安溪铁观音轻“发酵”做青工艺与品质特征的探讨[J].中国茶叶加工,2003,(1):21-22.
[21]冯廷佺."清香型"乌龙茶与"浓香型"乌龙茶之比较[J].中国茶叶,2005,27(5):18-19.
[22]高水练,林郑和,郝志龙,等.新旧工艺制成铁观音的品质化学比较[J].茶叶科学技术,2004,(3):27-28.
[23]陈晓栋,黄伙水.安溪铁观音的传统加工工艺及要点[J].福建茶叶,2009,(2):20-23.
[24]陈志雄,张稚秀,林室佳.传统制法与现代制法对乌龙茶品质影响分析[J].福建茶叶,2006,(3):14-16.
[25]姚珊珊.茶叶香气化学研究——茶叶及香花的香气化学[D].浙江大学,硕士学位论文,2005:23-27.
[26]钟秋生,吕海鹏,林智,等.东方美人茶和铁观音香气成分的比较研究[J].食品科学,2009,30(8):182-186.
[27]原利男.关于茶的香气成分(2)-乌龙茶的香气成分[J].黄华涛,译.福建茶叶,1986(1):14-15.
[28]胡海涛,苗爱清.乌龙茶香气研究进展[J].广东茶业,2002(5):10-14.
[29]周玲.乌龙茶香气挥发性成分及其感官性质分析[D].西南大学,硕士学位论文,2006-6-7.
[30]李凤凤,郭雯飞.碧螺珍珠茶与铁观音的香气成分分析与比较[J].浙江大学学报(理学版),2009,36(5):557-560.
[31]张方舟,陈荣冰,黄福平,等.春兰及其母本铁观音乌龙茶香气组分分析简报[J].福建农业学报,1999,14(2):33-36.
[32]游小青.茶树杂交后代及其亲本的香气特性及香型评价[J].中国茶叶,1993(1):26-28.
[33]丁勇.茶叶中糖苷酶类的研究进展[J].中国茶叶加工,2001,(4):34-36.
[34]Takeo T.,et al.Phytochemystry,1981,20(9):2149-2151.
[35]Sakata K.et al.,Proc.Symp.on Plant Growth and Enviroment Sawnkorea.1993:72-83.
[36]汪东风.茶叶中糖苷酶化学及其研究进展[J].中国茶叶加工,1997(4):32-36.
[37]Morita,K.et a1.Biosci.Biotech.Biochem.1994,58:687-690.
[38]Yano，M．et a1．Agric．Bio1．Chem．1991．55：1205-1806.
[39]Kobayashi．A．Biosci．Biotech．Biochem.1994．58:592-593.
[40]Sakaka,K.et a1.Agric.Bio1.Chem.1989,59,2975-2979.
[41]Guo W，Sakata K，Watnabe N.et al.Ceranyl-6-o-β-D-xylopranosyl-β-D-glucepyraneside isolated asaroma precursor from tea leaves for oolong tea.Phytochern,1993,33:1373-1375.
[42]Guo W,Hosoi R,Sakata K et al.(S)-Linalyl,2-phenylethyl,and benzyl disaccharide glycosides isulated asaroma precursors from oolong tea leaves.Biosci Biotech Biochern,1994,58(8):1532-1534.
[43]Moon JH,Watanabe N,Ijirna Y et al.Cis-and trans-lingalool3,7-oxides and methyl salicylate glycosidesand (Z)-3-hexenyl β-D-glucepyraneside as aroma precursors from tea leaves for oolong tea BiosciBiotech,1996,60(11):1815-1819.
[44]SakataK,Guo W,Moon JH.Tea chemistry.PartⅡ,With special reference to tea aroma precursors.In:JalnNK(ed).Glohd Advaflces in Tea Science.New Delhi:Aravail Books Inteanationa1.1999.692-704.
[45]Mizutani M,Nakanishi H,et a1.Cloning of B-primeverosidases from tea leaves,a key enzyme in tea aromaformation[M].Plant Physiol,2002,130:2164-2176.
[46]Ryals JA,Neuenschwander UH,et a1.Systemic acquired resistance.Plant Cell,1996,8:1809-1819.
[47]Partnaik S,Subramanyam VR,et a1.Antibacterial and antifungal activity of arom atic activity of aromaticconstituents of essential oils,M icrobios,1997,89:39-46.
[48]Mattiacci I,Dicke M,et a1.B-Glucosidases：all elicitor of herbivore-induced plant odor that attractshost-searching parasitic wasps.Proc Natl Acad Sci USA,1995,92:2036-2040.
[49]Pichersky E,Gershenzon J.The formation and function of plant volatiles:perfumes for pollinator attractionand defense.Curropin Plant Biol,2002,5:237-243.
[50]王华夫,游小青.茶叶中β-葡萄糖苷酶活性的测定[J].中国茶叶,1996(3):16-17.
[51]陈向东,藤尾雄策.日本根霉IF05318胞外β-葡萄糖苷酶的纯化及部分特性[J].微生物学报,1997,37(5):368-373.
[52]王迎春,李春华,刘晓军,等.茶叶葡萄糖苷酶研究进展[J].安徽农业科学,2008,36(10):4151-4153.
[53]宛晓春.水果风味及风味酶的研究[D].无锡:无锡轻工大学,1992.
[54]Ki Bong O H，Hamada K，Saito M．Isolation and properties of an extravellular β-glucosidase from afilamentous fungus,cladosponium resinae,isolated from kerosene [J].Biosci Biotech Biochem,l999,63(2):281-287.
[55]Guevas L,Niemeyer H M,Jonsson I M V．Partial purification and characterization of a hydroxamic acidglucoside β-D-glucosidase from maize [J].Phytochemistry,l992,l31(8):2609-2612.
[56]Takeo T．Production of linalool and geraniol by hydrolytic breakdown of bound forms in disrupted teashoots[J].Phytochemistry,l981.120(9):2l45-2147.
[57]江昌俊,李叶云,王朝霞.茶树鲜叶中β-葡萄糖苷酶提取条件的研究[J].南京农业大学学报,2000,23(2):93-96.
[58]骆耀平,董尚胜,童启庆,等.7个茶树品种新梢生育过程中β-葡萄糖苷酶活性变化[J].茶叶科学,l997,l7(增刊):l04-l07.
[59]夏涛,童启庆.冷冻萎凋对茶叶多酚氧化酶和β-葡萄糖苷酶活性影响初探[J].浙江农业大学学报,l997,23(1):l08-ll0.
[60]屠幼英,童启庆,骆耀平,等.茶叶香气释入机理研究——龙井茶炒制过程中β-葡萄糖苷酶和醇系香气的关系[J].茶叶,1999,25,(1):20-23.
[61]赵芹,童启庆.温肥对茶鲜叶β-葡萄糖苷酶活性及醇系香气的影响[J].浙江林学院学报,2000,l7(1):28-3l.
[62]张秀云.乌龙茶加工工艺与品质形成机理的研究[D].合肥:安徽农业大学,硕士学位论文,2000.
[63]张秀云,方世辉,夏涛.乌龙茶萎凋做青中β-葡萄糖苷酶活性变化研究[J].安徽农业大学学报,2000,27(2):164-166.
[64]Pandey S.Flavour-The queen of the characters I [J].The Assam Review and The News,1993,82(11/12):23-25/11-12.
[65]Pandey S.Flavour-The queen of the characters I [J].The Assam RevJew and The News,1994,83(1):9-11.
[66]赵和涛.茶多酚氧化酶研究及应用[J].茶叶科学技术,1996,(1):16-18.
[67]龙志荣,王登良,邱瑞瑾,等.水解酶对乌龙茶品质形成的影响[J].广东茶业,2004,(1):10-14.
[68]马春雷,陈亮.茶树功能基因分离克隆研究进展[J].分子植物育种,2006,4(3S):16-22.
[69]乔小燕,马春雷,陈亮.茶树黄酮合成酶Ⅱ基因全长cDNA序列的克隆和实时荧光定量PCR检测[J].茶叶科学,2009,29(5):347-354.
[70]马春雷,陈亮.茶树黄酮醇合成酶基因的克隆与原核表达[J].基因组学与应用生物学，2009,28(3):433-438.
[71]马春雷.茶树查尔酮异构酶、黄酮醇合成酶和无色花色素还原酶等基因的克隆与表达分析[D]中国农业科学院,硕士论文,2007-06-01.
[72]Devic M,Guilleminot J,Debeaujon I,et al.The BANYULS gene encodes a DFR-like protein and is amarker of early seed coat development[J].The Plant Journal,1999,19(4):387-398.
[73]胡波.茶树成花相关基因的cDNA克隆及生物信息学分析[D]福建农林大学,硕士论文,2010-04-01.
[74]李叶云,芦原坦.茶树咖啡碱生物合成相关基因表达研究[J].2008茶学青年科学家论坛论文集:187-192.
[75]Kato M,Mizuno K,Fujimura T.et al.Purification and characteriza-tion of caffeine synthase from tealeaves[J].Plant physiology.1999.120(2):579-586.
[76]李娟,邓婷婷,吴扬,等.茶氨酸合成酶基因的全长cDNA克隆及序列分析[J].茶叶学,2011,31(5):411-418.
[77]郭惠红,高述民,李凤兰,等.植物抗冻蛋白和抗寒基因表达的调控[J].植物生理学通讯,2003,39(6):555-560.
[78]Stockinger E J,Mao Y P,Regier M K,et al.Transcriptional adaptor and histone acetyltransferase proteinsin Arabidopsis and their interactions with CBF1,a transcriptional activator involved in cold-regulatedgene expression[J].Nucleic Acids Res,2001,29(7):1524~1533.
[79]房婉萍,邹中伟,侯喜林,等.茶树冷胁迫诱导H1-histone基因的克隆与序列分析张定[J].西北植物学报,2009,29(8):1514-1519.
[80]ELIZABETH A B,TUNG-YUAN S,MEENA S,et al.Water-deficit induction of a tomato H1-histonerequires abscisic acid[J].Plant Growth Regulation,1999,29(3):35-46.
[81]孙云.茶叶抗坏血酸过氧化物酶（APX）的生理学与分子生物学研究[D].福建农林大学,博士论文,2009-04-01.
[82]张亚丽.茶树ACS、ACO基因克隆与亲环素基因的鉴定及其表达分析[D].中国农业科学院,硕士论文,2007-06-01.
[83]王朝霞,李叶云,江昌俊,等,茶树巯基蛋白酶抑制剂基因的cDNA克隆与序列分析[J].茶叶科学,2005,25(3):177-182.
[84]李远华,江昌俊,杨顺利,等.β-葡萄糖苷酶cDNA克隆和原核表达[J].农业生物技术学报,2004,12(6):625-629.
[85]Mizutani M.,Nakanishi H.,Ema J.,Ma S.J.,Noguchi E.,Ino-hara-Ochiai M.,Fukuchi-Mizutani M.,NakaoM.,and Saka-ta K.,2002,Cloning ofβ-primeverosidase from tea leaves,akey enzyme in tea aromaformation,Plant Physiol.,130:2164-2176.
[86]赵丽萍,陈亮,王新超,等.茶树新稍不同叶片中β-葡萄糖苷酶和β-樱草糖苷酶基因表达的实时定量PCR分析,茶叶科学,2006,26(1):11-16.
[87]王让剑,江昌俊,张正竹,等.两种茶树β-葡萄糖苷酶基因cDNA在原核中表达的研究[J].生物技术通报,2007,(4),:102-105.
[88]张广辉,茶树遗传转化及UV-B对香气相关基因表达影响的研究[D]浙江大学农业,博士论文,2007-01.
[89]福建安溪涉茶产业年产值达73亿元[EB/OL].新华网.http://news.163.com/10/1114/10/6LEML0QA00014JB5.html.2010-11-14.
[90]王欢,张振民.影响茶叶香气的几种因素[J].福建茶叶.1997,(3):16-22.
[91]何孝延.乌龙茶香气成分形成及其影响因素[J].茶叶科学简报,1994,(4):5-8.
[92]王日为,张丽霞,杨伟丽,等.乌龙茶做青过程中香气的动态变化规律[J].湖南农业大学学报.1994,(3):194-199.
[93]张婉婷,张灵枝,王登良.加工工艺对乌龙茶香气成分影响的研究进展[J].中国茶叶,2010,(4):10-13.
[94]苗爱清,吕海鹏,孙世利,等.乌龙茶香气的HS-SPME-GC-MS/GC-O研究[J].茶叶科学,2010,30(增刊1):583-587.
[95]张冉.α-法尼烯合成酶基因转化拟南芥和烟草的研究[D].山东:山东农业大学,硕士学位论文,2011-06.
[96]百度百科[EB/OL].百度网.http://baike.baidu.com/view/702939.htm.2010-06-26
[97]李冬梅,赵振东,孙震,等.香叶基丙酮合成反应馏出物组成及形成机理探讨[J].林产化工通讯,2003,37(6):8-11.
[98]单海霞,陆杨,李在均,等.新型温控离子液体介质中脂肪酶催化合成乙酸苯乙酯[J].催化学报,2010,31:289-294.
[99]闫栋.钯催化的多取代吲哚合成研究[D].上海:上海大学,硕士论文,2010-05.
[100]任浩明,吕秀阳.含氨高温液态水中苯乙腈水解反应动力学[J].化工学报,2009,60(6):1435-1441.
[101] Farmer E E.Surface-to-air signals.Nature,2001,411:854-856.
[102] Arimura G,Ozawa R,Shimoda T,et al.Herbivory-induced volatiles elicit defense genes in lima beanleaves.Nature,2000,406:512-515.
[103]叶琼.生物法拆分制备L-薄荷醇[D].浙江:浙江大学材化学院,硕士学位论文,2008-05.
[104]何建英,刘素环,陈丹云,等.己酸己酯的催化合成[J].企业科技与发展,2007,17:47-48.
[105]桂连友,刘树生,陈宗懋.外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理[J].昆虫学报,2004,47(4):507-514.
[106]竹尾忠一等(梁月荣译).乌龙茶和红茶香气的食品化学研究[J].福建茶叶,1986,(3):40-42.
[107]竹尾忠一.乌龙茶香气及其特征[J].国外农学茶叶,1984,(4):16-22.
[108]AKio Kobayashi等,周巨根译.日光萎凋和室内萎调期间摇青处理对包种茶香气形成的影响[J].福建茶叶,1986,(2):44-48.
[109]杨贤强,沈生荣.人工光照对离体茶鲜叶氨基酸及香气成分的影响[J].福建茶叶,1991,(1):11-21.
[110]王日为,张丽霞,杨伟丽,等.乌龙茶做青过程中香气的动态变化规律[J].湖南农业大学学报,1999,25(3):194-199.
[111]苗爱清,伍锡岳,庞式,等.岭头单丛茶晒青香气变化研究[J].广东茶业,2001,(4):35-38.
[112]叶乃兴.乌龙茶的香气成分[J].茶叶科学简报,1990.(2):1-5.
[113]林正奎,华映芳,谷豫红,等.茶鲜叶挥发油化学成分研究[J].植物学报,1982,25(5):440-449.
[114]骆少君,濮荷娟,郭雯飞.不同品种乌龙茶香气的特征及其与品质等级的相关性[J].福建茶叶,1987,(2):11-20.
[115]陈荣冰,张方舟,黄福平.丹桂与名优乌龙茶品种香气特征比较[J].茶叶科学,1998,18(2):113-118.
[116]张方舟,陈荣冰,黄福平.春兰及其母本铁观音乌龙茶香气组分分析简报[J].福建农业学报,1998,14(2):33-36.
[117]AKio Kobayashi等,周巨根译.日光萎凋和室内萎调期间摇青处理对包种茶香气形成的影响[J].福建茶叶,1986,(2):44-48.
[118]黄福平.乌龙茶(Camellia Sinensis L.)做青过程中生理生化的变化及其香气形成[D].福建农林大学,硕士学位论文,2001-05.
[119]钟萝主编.茶叶品质理化分析[M].上海:上海科学技术出版社.1989.12.
[120]王日为,张丽霞,杨伟丽,等.乌龙茶做青过程中香气的动态变化规律[J].湖南农业大学学报,1999,25(3):194-199.
[121]苗爱清,伍锡岳,庞式,等.岭头单丛茶晒青香气变化研究[J].广东茶业,2001,(4):35-38.
[122]周昱,魏宏,吴天送,等.乌龙茶“铁观音”香气成分的气相色谱/质谱分析[J].色谱,1994,12(5):355-357.
[123]张木树,乌龙茶香气与品质审评[J].福建茶叶,1998,(2):43-45.
[124]何孝延.乌龙茶香气成分形成及其影响因素[J].茶叶科技简报,1994,(4):5-8.
[125]陈志雄,张稚秀.传统制法与现代制法对乌龙茶品质的影响[J].中国茶叶,2006,5:33-34.
[126]金心怡,孙云,孙威江,等.清香型乌龙茶品质特征与发展现状[J].中国茶叶,2007,1:12-13.
[127]杨慧君,姚娜,李潞滨,等.建兰花香成分的GC-MS分析[J].中国农学通报,2011,27(16):104-109.
[128]刘扬岷,王利平,袁身淑.固相微萃取气质联用分析白兰花的香气成分[J].无锡轻工业大学学报,2001,20(4):427-429.
[129]张晓林,林祖铭,金声,等.杭州桂花头香成分的研究[J].高等学校化学学报,1986,7(8):695-700.
[130]祝美莉,丁德生,黄祖萱,等.桂花不同变种的头香成分研究[J].植物学报,1985,27(4):412-418.
[131]冯建跃,赵菁,黄巧巧.吸附丝色谱-质谱法用于桂花香气研究[J].浙江大学学报(理学版),2001,28(6):672-675.
[132]田光辉,刘存芳,辜天琪,等.衰弱桂花香气成分的GC-MS分析[J].安徽农业大学,2008,36(17):7214-7216.
[133]金荷仙,陈俊愉,金幼菊.南京不同类型梅花品种香气成分的比较研究[J].园艺学报,2005,(6):1139.
[134]陈青,姚蓉君,张前军.固相微萃取气质联用分析野茉莉花的香气成分[J].精细化工,2007,24(2):159-161.
[135]赵喜兰,常陆林,任丽平.栀子花挥发油的GC-MS的指纹图谱[J].安徽农业科学,2009,37(8):3355-3356.
[136]张晓萌.桃果实成熟过程中香气成分形成及其生机制研究[J].浙江大学,硕士学位论文,2005-05-01.
[137]韦节华.肥城桃香气组成及其影响因素研究[J].山东农业大学,硕士学位论文,2008-06-13.
[138]魏树伟.套袋苹果贮藏生理及香气变化研究[J].山东农业大学,硕士学位论文,2008-6-16.
[139]赵和涛.茶多酚氧化酶研究及应用[J].茶叶科学技术,1996,(1):16-18.
[140]禹利君.乌龙茶加工中氧化还原酶的活性研究[D].长沙：湖南农业大学硕士学位论文,1998
[141]唐颢,杨伟丽,文海涛.乌龙茶新工艺做青期间果胶酶的活性变化及其生化效应研究[J].茶叶科学,2005,25(3):197-202.
[142]王若仲,杨伟丽,禹利君.乌龙茶中加工中蛋白酶活性与相关生化成分的变化[J].茶叶科学,2001,21(1):30-34.
[143]唐颢,杨伟丽,文海涛.乌龙茶加工中纤维素酶的活性与相关生化成分的动态变化[J].茶叶通讯,2003,(2):19-21.
[144]王若仲,杨伟丽,禹利君.乌龙茶加工中淀粉酶活性与相关生化成分变化研究[J].茶叶科学,2002,22(1):83-86.
[145]Takeo T,et a1.The general directory of tea processing plants[M].Japan.1987.128-131.
[146]Takeo T.Production of linalol and geraniol by hydrolytic breakdown of hound forms in disrupted teashoots[J].Phytochemistry.1981.20:2145-2147.
[147]Sakata K,Guo W,Hosoi R,et al.Studies on aroma formation mechanism of ooiong tea:Isolation ofglycosidic aroma precursors and characterization of the enzymes involved in the aromaformation.In:Proc Symp on Plant Growth and Environment[c].Korea,1993,73-83.
[148]杨锐. β-葡萄糖苷酶对暑秋季新工艺乌龙茶品质影响研究[D].福州:福建农林大学,硕士学位论文,2007-04.
[149]骆耀平,董尚胜,童启庆,等.7个茶树品种新梢生育过程中葡萄糖苷酶活性变化[J].茶叶学,1997,17(增):104-107.
[150]张正竹,宛晓春,施兆鹏,等.鲜茶叶摊放过程中呼吸速率、葡萄糖苷酶活性、游离态香气和糖苷类香气前体含量的变化[J].植物生理学通讯,2003,39(2):134-136.
[151]屠幼英,童启庆,骆耀平,等.龙井茶叶加工过程中醇系香气释放机理研究[J].浙江大学学报,1999,25(5):535.
[152]刘莉华,宛晓春,文勇,等.祁门红茶加工过程中葡萄糖苷酶活性变化研究[J].安徽农业大学学报,2003,30(4):386-389.
[153]夏涛,童启庆,董尚胜,等.红茶萎凋发酵中葡萄糖苷酶的活性变化[J].茶叶科学,1996,16(1):63-66.
[154]赵东,刘祖生,奚彪.茶树多酚氧化酶基因的克隆及其序列比较[J].茶叶科学,21(2):94-98
[155]马春雷.茶树查尔酮异构酶、黄酮醇合成酶和无色花色素还原酶等基因的克隆与表达分析[D]中国农业科学院,硕士论文,2007-06-01.
[156]李远华,江昌俊,宛晓春.茶树咖啡碱合成酶基因mRNA表达的研究[J].茶叶科学，2004，21(1):23-28.
[157]Sugiyama T.,and Sadzuka Y.,1999,Combination of theaninewith doxorubicin inhibits hepatic metastasisof M5076ovar-ian sarcoma,Clinical Cancer Res.,5(2):413-416.
[158]陈暄,房婉萍,邹中伟,等.茶树冷胁迫诱导抗寒基因CBF的克隆与表达分析[J].茶叶科学2009,29(1):53-59.
[159]张亚丽.茶树ACS、ACO基因克隆与亲环素基因的鉴定及其表达分析[D].中国农业科学院,硕士论文,2007-06-01.
[160]刑浩然,刘丽娟,刘国振.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21:1-6.
[161]汤文文. BRSK2对能量压力诱导下细胞周期适应性变化的调控作用研究[D].上海:复旦大学,博士学位论文,2006-11-20.
[162]李远华,江昌俊,杨顺利,等.茶树葡萄糖苷酶cDNA克隆和原核表达[J].农业生物技术学报,2004,12(6):625-629.
[163]马振刚,唐婧,马淑华,等.新型碱性低分子量β-葡萄糖苷酶基因的分离与酶学性质研究[J].食品工业科技,2012,(4):108-113.
[164]唐婧.一株产纤维素酶细菌的分离及其β-葡萄糖苷酶基因克隆[D].西南大学,硕士学位论文,2008.
[165]张正竹.绿茶主要香气物质的糖昔类前体研究[D].湖南农业大学,博士学位论文,2000.
[166]李远华.茶树β-葡萄糖苷酶基因克隆、表达和分布[D].安徽农业大学,博士学位论文,2003-10.
[167] Haller F,Kulle B,Schwager S,et al,Equivalence test in quantitative reverse transcription polymerasechain reaction:confirmation of refernce genes suitable for normalization.Analytixal Biochemistry,2004,335:1-9.
[168]张珣.六种植物病毒Real Time PCR定量方法的建立及应用[D].北京:中国农业科学院.博士论文,2008.
[169]洪云,李津,汪和睦,等.实时荧光定量PCR技术进展[J].国际流行病学传染病学杂志,2006,33(3):161-166.
[170]高斌,刘敬忠.实时荧光PCR技术的研究及其应用进展[J].诊断学理论与实践,2005,4(6):507-509.
[2]周昱,魏宏,吴天送,等.乌龙茶“铁观音”香气成分的气相色谱/质谱分析[J].色谱,1994,12(3):355-357.
[3]于欣洋,岳文杰,李金辉,等.茶叶香气研究进展[J].茶叶科学技术,2008.(3):9-13.
[4]陈美霞,陈学森,周杰.蒸馏-萃取法与溶剂萃取法提取杏果实香气成分的比较[J].分析试验室,2005,24(3):65-70.
[5]宛晓春.茶叶生物化学[M].北京:中国农业出版社,2003.8:39-49.
[6]林正奎,华映芳,谷豫红,等.茶鲜叶挥发油化学成分的研究[J].植物学报,1982,124(2):23-28.
[7]吴勇.萜烯类化合物与茶叶香气[J].化学工程与装备,2009,(11):123-125.
[8]张长波,孙红霞,巩中军,等.植物萜类化合物的天然合成途径及其相关合酶[J].植物生理学通讯,2007,43(4):779-785.
[9]吕连梅,董尚胜.茶叶香气的研究进展[J].茶叶,2002,28(4):181-184.
[10]朱旗,施兆鹏,任春梅.绿茶香气不同提取方法的研究[J].茶叶科学,2001,21(l):38-43.
[11]孙宗保,赵杰文,邹小波,等.HS-SPME/GC-MS/GC-O对镇江香醋特征香气成分的确定[J].江苏大学学报,2010,31(2):139-144.
[12]马永昆,蒋家奎,魏永义,等.基于GC-MS与嗅闻联用的镇江香醋香气指纹图谱研究[J].食品科学,2007,28(9):496-499.
[13]夏玲君,宋焕禄.香味检测技术GC-O的应用[J].食品与发酵工业,2006,32(1):83-87.
[14]吉克温,金心怡,苏益平.乌龙茶空调制茶技术要点[J].福建茶叶,2004,(1):16.
[15]郑乃辉,姚信恩,林锦江,等.轻发酵乌龙茶工艺技术总结[J].茶叶科学技术,2004,(2):34,41.
[16]谢萍娟,李旭云.清香型铁观音的制作技术[J].中国茶叶,2006,(4):32-33.
[17]孙云,吉克温,杨江帆,等.清香型乌龙茶加工技术与配套设备[J].中国茶叶,2007,(3):9-11.
[18]孙云,吉克温,杨江帆,等.清香型乌龙茶加工技术与配套设备(续)[J].中国茶叶,2007,(4):8-10.
[19]张方舟,陈加友.清香型乌龙茶空调做青设备及加工的关键技术[J].中国茶叶,2005,(1):32-33.
[20]李宗恒,陈郁榕.安溪铁观音轻“发酵”做青工艺与品质特征的探讨[J].中国茶叶加工,2003,(1):21-22.
[21]冯廷佺."清香型"乌龙茶与"浓香型"乌龙茶之比较[J].中国茶叶,2005,27(5):18-19.
[22]高水练,林郑和,郝志龙,等.新旧工艺制成铁观音的品质化学比较[J].茶叶科学技术,2004,(3):27-28.
[23]陈晓栋,黄伙水.安溪铁观音的传统加工工艺及要点[J].福建茶叶,2009,(2):20-23.
[24]陈志雄,张稚秀,林室佳.传统制法与现代制法对乌龙茶品质影响分析[J].福建茶叶,2006,(3):14-16.
[25]姚珊珊.茶叶香气化学研究——茶叶及香花的香气化学[D].浙江大学,硕士学位论文,2005:23-27.
[26]钟秋生,吕海鹏,林智,等.东方美人茶和铁观音香气成分的比较研究[J].食品科学,2009,30(8):182-186.
[27]原利男.关于茶的香气成分(2)-乌龙茶的香气成分[J].黄华涛,译.福建茶叶,1986(1):14-15.
[28]胡海涛,苗爱清.乌龙茶香气研究进展[J].广东茶业,2002(5):10-14.
[29]周玲.乌龙茶香气挥发性成分及其感官性质分析[D].西南大学,硕士学位论文,2006-6-7.
[30]李凤凤,郭雯飞.碧螺珍珠茶与铁观音的香气成分分析与比较[J].浙江大学学报(理学版),2009,36(5):557-560.
[31]张方舟,陈荣冰,黄福平,等.春兰及其母本铁观音乌龙茶香气组分分析简报[J].福建农业学报,1999,14(2):33-36.
[32]游小青.茶树杂交后代及其亲本的香气特性及香型评价[J].中国茶叶,1993(1):26-28.
[33]丁勇.茶叶中糖苷酶类的研究进展[J].中国茶叶加工,2001,(4):34-36.
[34]Takeo T.,et al.Phytochemystry,1981,20(9):2149-2151.
[35]Sakata K.et al.,Proc.Symp.on Plant Growth and Enviroment Sawnkorea.1993:72-83.
[36]汪东风.茶叶中糖苷酶化学及其研究进展[J].中国茶叶加工,1997(4):32-36.
[37]Morita,K.et a1.Biosci.Biotech.Biochem.1994,58:687-690.
[38]Yano，M．et a1．Agric．Bio1．Chem．1991．55：1205-1806.
[39]Kobayashi．A．Biosci．Biotech．Biochem.1994．58:592-593.
[40]Sakaka,K.et a1.Agric.Bio1.Chem.1989,59,2975-2979.
[41]Guo W，Sakata K，Watnabe N.et al.Ceranyl-6-o-β-D-xylopranosyl-β-D-glucepyraneside isolated asaroma precursor from tea leaves for oolong tea.Phytochern,1993,33:1373-1375.
[42]Guo W,Hosoi R,Sakata K et al.(S)-Linalyl,2-phenylethyl,and benzyl disaccharide glycosides isulated asaroma precursors from oolong tea leaves.Biosci Biotech Biochern,1994,58(8):1532-1534.
[43]Moon JH,Watanabe N,Ijirna Y et al.Cis-and trans-lingalool3,7-oxides and methyl salicylate glycosidesand (Z)-3-hexenyl β-D-glucepyraneside as aroma precursors from tea leaves for oolong tea BiosciBiotech,1996,60(11):1815-1819.
[44]SakataK,Guo W,Moon JH.Tea chemistry.PartⅡ,With special reference to tea aroma precursors.In:JalnNK(ed).Glohd Advaflces in Tea Science.New Delhi:Aravail Books Inteanationa1.1999.692-704.
[45]Mizutani M,Nakanishi H,et a1.Cloning of B-primeverosidases from tea leaves,a key enzyme in tea aromaformation[M].Plant Physiol,2002,130:2164-2176.
[46]Ryals JA,Neuenschwander UH,et a1.Systemic acquired resistance.Plant Cell,1996,8:1809-1819.
[47]Partnaik S,Subramanyam VR,et a1.Antibacterial and antifungal activity of arom atic activity of aromaticconstituents of essential oils,M icrobios,1997,89:39-46.
[48]Mattiacci I,Dicke M,et a1.B-Glucosidases：all elicitor of herbivore-induced plant odor that attractshost-searching parasitic wasps.Proc Natl Acad Sci USA,1995,92:2036-2040.
[49]Pichersky E,Gershenzon J.The formation and function of plant volatiles:perfumes for pollinator attractionand defense.Curropin Plant Biol,2002,5:237-243.
[50]王华夫,游小青.茶叶中β-葡萄糖苷酶活性的测定[J].中国茶叶,1996(3):16-17.
[51]陈向东,藤尾雄策.日本根霉IF05318胞外β-葡萄糖苷酶的纯化及部分特性[J].微生物学报,1997,37(5):368-373.
[52]王迎春,李春华,刘晓军,等.茶叶葡萄糖苷酶研究进展[J].安徽农业科学,2008,36(10):4151-4153.
[53]宛晓春.水果风味及风味酶的研究[D].无锡:无锡轻工大学,1992.
[54]Ki Bong O H，Hamada K，Saito M．Isolation and properties of an extravellular β-glucosidase from afilamentous fungus,cladosponium resinae,isolated from kerosene [J].Biosci Biotech Biochem,l999,63(2):281-287.
[55]Guevas L,Niemeyer H M,Jonsson I M V．Partial purification and characterization of a hydroxamic acidglucoside β-D-glucosidase from maize [J].Phytochemistry,l992,l31(8):2609-2612.
[56]Takeo T．Production of linalool and geraniol by hydrolytic breakdown of bound forms in disrupted teashoots[J].Phytochemistry,l981.120(9):2l45-2147.
[57]江昌俊,李叶云,王朝霞.茶树鲜叶中β-葡萄糖苷酶提取条件的研究[J].南京农业大学学报,2000,23(2):93-96.
[58]骆耀平,董尚胜,童启庆,等.7个茶树品种新梢生育过程中β-葡萄糖苷酶活性变化[J].茶叶科学,l997,l7(增刊):l04-l07.
[59]夏涛,童启庆.冷冻萎凋对茶叶多酚氧化酶和β-葡萄糖苷酶活性影响初探[J].浙江农业大学学报,l997,23(1):l08-ll0.
[60]屠幼英,童启庆,骆耀平,等.茶叶香气释入机理研究——龙井茶炒制过程中β-葡萄糖苷酶和醇系香气的关系[J].茶叶,1999,25,(1):20-23.
[61]赵芹,童启庆.温肥对茶鲜叶β-葡萄糖苷酶活性及醇系香气的影响[J].浙江林学院学报,2000,l7(1):28-3l.
[62]张秀云.乌龙茶加工工艺与品质形成机理的研究[D].合肥:安徽农业大学,硕士学位论文,2000.
[63]张秀云,方世辉,夏涛.乌龙茶萎凋做青中β-葡萄糖苷酶活性变化研究[J].安徽农业大学学报,2000,27(2):164-166.
[64]Pandey S.Flavour-The queen of the characters I [J].The Assam Review and The News,1993,82(11/12):23-25/11-12.
[65]Pandey S.Flavour-The queen of the characters I [J].The Assam RevJew and The News,1994,83(1):9-11.
[66]赵和涛.茶多酚氧化酶研究及应用[J].茶叶科学技术,1996,(1):16-18.
[67]龙志荣,王登良,邱瑞瑾,等.水解酶对乌龙茶品质形成的影响[J].广东茶业,2004,(1):10-14.
[68]马春雷,陈亮.茶树功能基因分离克隆研究进展[J].分子植物育种,2006,4(3S):16-22.
[69]乔小燕,马春雷,陈亮.茶树黄酮合成酶Ⅱ基因全长cDNA序列的克隆和实时荧光定量PCR检测[J].茶叶科学,2009,29(5):347-354.
[70]马春雷,陈亮.茶树黄酮醇合成酶基因的克隆与原核表达[J].基因组学与应用生物学，2009,28(3):433-438.
[71]马春雷.茶树查尔酮异构酶、黄酮醇合成酶和无色花色素还原酶等基因的克隆与表达分析[D]中国农业科学院,硕士论文,2007-06-01.
[72]Devic M,Guilleminot J,Debeaujon I,et al.The BANYULS gene encodes a DFR-like protein and is amarker of early seed coat development[J].The Plant Journal,1999,19(4):387-398.
[73]胡波.茶树成花相关基因的cDNA克隆及生物信息学分析[D]福建农林大学,硕士论文,2010-04-01.
[74]李叶云,芦原坦.茶树咖啡碱生物合成相关基因表达研究[J].2008茶学青年科学家论坛论文集:187-192.
[75]Kato M,Mizuno K,Fujimura T.et al.Purification and characteriza-tion of caffeine synthase from tealeaves[J].Plant physiology.1999.120(2):579-586.
[76]李娟,邓婷婷,吴扬,等.茶氨酸合成酶基因的全长cDNA克隆及序列分析[J].茶叶学,2011,31(5):411-418.
[77]郭惠红,高述民,李凤兰,等.植物抗冻蛋白和抗寒基因表达的调控[J].植物生理学通讯,2003,39(6):555-560.
[78]Stockinger E J,Mao Y P,Regier M K,et al.Transcriptional adaptor and histone acetyltransferase proteinsin Arabidopsis and their interactions with CBF1,a transcriptional activator involved in cold-regulatedgene expression[J].Nucleic Acids Res,2001,29(7):1524~1533.
[79]房婉萍,邹中伟,侯喜林,等.茶树冷胁迫诱导H1-histone基因的克隆与序列分析张定[J].西北植物学报,2009,29(8):1514-1519.
[80]ELIZABETH A B,TUNG-YUAN S,MEENA S,et al.Water-deficit induction of a tomato H1-histonerequires abscisic acid[J].Plant Growth Regulation,1999,29(3):35-46.
[81]孙云.茶叶抗坏血酸过氧化物酶（APX）的生理学与分子生物学研究[D].福建农林大学,博士论文,2009-04-01.
[82]张亚丽.茶树ACS、ACO基因克隆与亲环素基因的鉴定及其表达分析[D].中国农业科学院,硕士论文,2007-06-01.
[83]王朝霞,李叶云,江昌俊,等,茶树巯基蛋白酶抑制剂基因的cDNA克隆与序列分析[J].茶叶科学,2005,25(3):177-182.
[84]李远华,江昌俊,杨顺利,等.β-葡萄糖苷酶cDNA克隆和原核表达[J].农业生物技术学报,2004,12(6):625-629.
[85]Mizutani M.,Nakanishi H.,Ema J.,Ma S.J.,Noguchi E.,Ino-hara-Ochiai M.,Fukuchi-Mizutani M.,NakaoM.,and Saka-ta K.,2002,Cloning ofβ-primeverosidase from tea leaves,akey enzyme in tea aromaformation,Plant Physiol.,130:2164-2176.
[86]赵丽萍,陈亮,王新超,等.茶树新稍不同叶片中β-葡萄糖苷酶和β-樱草糖苷酶基因表达的实时定量PCR分析,茶叶科学,2006,26(1):11-16.
[87]王让剑,江昌俊,张正竹,等.两种茶树β-葡萄糖苷酶基因cDNA在原核中表达的研究[J].生物技术通报,2007,(4),:102-105.
[88]张广辉,茶树遗传转化及UV-B对香气相关基因表达影响的研究[D]浙江大学农业,博士论文,2007-01.
[89]福建安溪涉茶产业年产值达73亿元[EB/OL].新华网.http://news.163.com/10/1114/10/6LEML0QA00014JB5.html.2010-11-14.
[90]王欢,张振民.影响茶叶香气的几种因素[J].福建茶叶.1997,(3):16-22.
[91]何孝延.乌龙茶香气成分形成及其影响因素[J].茶叶科学简报,1994,(4):5-8.
[92]王日为,张丽霞,杨伟丽,等.乌龙茶做青过程中香气的动态变化规律[J].湖南农业大学学报.1994,(3):194-199.
[93]张婉婷,张灵枝,王登良.加工工艺对乌龙茶香气成分影响的研究进展[J].中国茶叶,2010,(4):10-13.
[94]苗爱清,吕海鹏,孙世利,等.乌龙茶香气的HS-SPME-GC-MS/GC-O研究[J].茶叶科学,2010,30(增刊1):583-587.
[95]张冉.α-法尼烯合成酶基因转化拟南芥和烟草的研究[D].山东:山东农业大学,硕士学位论文,2011-06.
[96]百度百科[EB/OL].百度网.http://baike.baidu.com/view/702939.htm.2010-06-26
[97]李冬梅,赵振东,孙震,等.香叶基丙酮合成反应馏出物组成及形成机理探讨[J].林产化工通讯,2003,37(6):8-11.
[98]单海霞,陆杨,李在均,等.新型温控离子液体介质中脂肪酶催化合成乙酸苯乙酯[J].催化学报,2010,31:289-294.
[99]闫栋.钯催化的多取代吲哚合成研究[D].上海:上海大学,硕士论文,2010-05.
[100]任浩明,吕秀阳.含氨高温液态水中苯乙腈水解反应动力学[J].化工学报,2009,60(6):1435-1441.
[101] Farmer E E.Surface-to-air signals.Nature,2001,411:854-856.
[102] Arimura G,Ozawa R,Shimoda T,et al.Herbivory-induced volatiles elicit defense genes in lima beanleaves.Nature,2000,406:512-515.
[103]叶琼.生物法拆分制备L-薄荷醇[D].浙江:浙江大学材化学院,硕士学位论文,2008-05.
[104]何建英,刘素环,陈丹云,等.己酸己酯的催化合成[J].企业科技与发展,2007,17:47-48.
[105]桂连友,刘树生,陈宗懋.外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理[J].昆虫学报,2004,47(4):507-514.
[106]竹尾忠一等(梁月荣译).乌龙茶和红茶香气的食品化学研究[J].福建茶叶,1986,(3):40-42.
[107]竹尾忠一.乌龙茶香气及其特征[J].国外农学茶叶,1984,(4):16-22.
[108]AKio Kobayashi等,周巨根译.日光萎凋和室内萎调期间摇青处理对包种茶香气形成的影响[J].福建茶叶,1986,(2):44-48.
[109]杨贤强,沈生荣.人工光照对离体茶鲜叶氨基酸及香气成分的影响[J].福建茶叶,1991,(1):11-21.
[110]王日为,张丽霞,杨伟丽,等.乌龙茶做青过程中香气的动态变化规律[J].湖南农业大学学报,1999,25(3):194-199.
[111]苗爱清,伍锡岳,庞式,等.岭头单丛茶晒青香气变化研究[J].广东茶业,2001,(4):35-38.
[112]叶乃兴.乌龙茶的香气成分[J].茶叶科学简报,1990.(2):1-5.
[113]林正奎,华映芳,谷豫红,等.茶鲜叶挥发油化学成分研究[J].植物学报,1982,25(5):440-449.
[114]骆少君,濮荷娟,郭雯飞.不同品种乌龙茶香气的特征及其与品质等级的相关性[J].福建茶叶,1987,(2):11-20.
[115]陈荣冰,张方舟,黄福平.丹桂与名优乌龙茶品种香气特征比较[J].茶叶科学,1998,18(2):113-118.
[116]张方舟,陈荣冰,黄福平.春兰及其母本铁观音乌龙茶香气组分分析简报[J].福建农业学报,1998,14(2):33-36.
[117]AKio Kobayashi等,周巨根译.日光萎凋和室内萎调期间摇青处理对包种茶香气形成的影响[J].福建茶叶,1986,(2):44-48.
[118]黄福平.乌龙茶(Camellia Sinensis L.)做青过程中生理生化的变化及其香气形成[D].福建农林大学,硕士学位论文,2001-05.
[119]钟萝主编.茶叶品质理化分析[M].上海:上海科学技术出版社.1989.12.
[120]王日为,张丽霞,杨伟丽,等.乌龙茶做青过程中香气的动态变化规律[J].湖南农业大学学报,1999,25(3):194-199.
[121]苗爱清,伍锡岳,庞式,等.岭头单丛茶晒青香气变化研究[J].广东茶业,2001,(4):35-38.
[122]周昱,魏宏,吴天送,等.乌龙茶“铁观音”香气成分的气相色谱/质谱分析[J].色谱,1994,12(5):355-357.
[123]张木树,乌龙茶香气与品质审评[J].福建茶叶,1998,(2):43-45.
[124]何孝延.乌龙茶香气成分形成及其影响因素[J].茶叶科技简报,1994,(4):5-8.
[125]陈志雄,张稚秀.传统制法与现代制法对乌龙茶品质的影响[J].中国茶叶,2006,5:33-34.
[126]金心怡,孙云,孙威江,等.清香型乌龙茶品质特征与发展现状[J].中国茶叶,2007,1:12-13.
[127]杨慧君,姚娜,李潞滨,等.建兰花香成分的GC-MS分析[J].中国农学通报,2011,27(16):104-109.
[128]刘扬岷,王利平,袁身淑.固相微萃取气质联用分析白兰花的香气成分[J].无锡轻工业大学学报,2001,20(4):427-429.
[129]张晓林,林祖铭,金声,等.杭州桂花头香成分的研究[J].高等学校化学学报,1986,7(8):695-700.
[130]祝美莉,丁德生,黄祖萱,等.桂花不同变种的头香成分研究[J].植物学报,1985,27(4):412-418.
[131]冯建跃,赵菁,黄巧巧.吸附丝色谱-质谱法用于桂花香气研究[J].浙江大学学报(理学版),2001,28(6):672-675.
[132]田光辉,刘存芳,辜天琪,等.衰弱桂花香气成分的GC-MS分析[J].安徽农业大学,2008,36(17):7214-7216.
[133]金荷仙,陈俊愉,金幼菊.南京不同类型梅花品种香气成分的比较研究[J].园艺学报,2005,(6):1139.
[134]陈青,姚蓉君,张前军.固相微萃取气质联用分析野茉莉花的香气成分[J].精细化工,2007,24(2):159-161.
[135]赵喜兰,常陆林,任丽平.栀子花挥发油的GC-MS的指纹图谱[J].安徽农业科学,2009,37(8):3355-3356.
[136]张晓萌.桃果实成熟过程中香气成分形成及其生机制研究[J].浙江大学,硕士学位论文,2005-05-01.
[137]韦节华.肥城桃香气组成及其影响因素研究[J].山东农业大学,硕士学位论文,2008-06-13.
[138]魏树伟.套袋苹果贮藏生理及香气变化研究[J].山东农业大学,硕士学位论文,2008-6-16.
[139]赵和涛.茶多酚氧化酶研究及应用[J].茶叶科学技术,1996,(1):16-18.
[140]禹利君.乌龙茶加工中氧化还原酶的活性研究[D].长沙：湖南农业大学硕士学位论文,1998
[141]唐颢,杨伟丽,文海涛.乌龙茶新工艺做青期间果胶酶的活性变化及其生化效应研究[J].茶叶科学,2005,25(3):197-202.
[142]王若仲,杨伟丽,禹利君.乌龙茶中加工中蛋白酶活性与相关生化成分的变化[J].茶叶科学,2001,21(1):30-34.
[143]唐颢,杨伟丽,文海涛.乌龙茶加工中纤维素酶的活性与相关生化成分的动态变化[J].茶叶通讯,2003,(2):19-21.
[144]王若仲,杨伟丽,禹利君.乌龙茶加工中淀粉酶活性与相关生化成分变化研究[J].茶叶科学,2002,22(1):83-86.
[145]Takeo T,et a1.The general directory of tea processing plants[M].Japan.1987.128-131.
[146]Takeo T.Production of linalol and geraniol by hydrolytic breakdown of hound forms in disrupted teashoots[J].Phytochemistry.1981.20:2145-2147.
[147]Sakata K,Guo W,Hosoi R,et al.Studies on aroma formation mechanism of ooiong tea:Isolation ofglycosidic aroma precursors and characterization of the enzymes involved in the aromaformation.In:Proc Symp on Plant Growth and Environment[c].Korea,1993,73-83.
[148]杨锐. β-葡萄糖苷酶对暑秋季新工艺乌龙茶品质影响研究[D].福州:福建农林大学,硕士学位论文,2007-04.
[149]骆耀平,董尚胜,童启庆,等.7个茶树品种新梢生育过程中葡萄糖苷酶活性变化[J].茶叶学,1997,17(增):104-107.
[150]张正竹,宛晓春,施兆鹏,等.鲜茶叶摊放过程中呼吸速率、葡萄糖苷酶活性、游离态香气和糖苷类香气前体含量的变化[J].植物生理学通讯,2003,39(2):134-136.
[151]屠幼英,童启庆,骆耀平,等.龙井茶叶加工过程中醇系香气释放机理研究[J].浙江大学学报,1999,25(5):535.
[152]刘莉华,宛晓春,文勇,等.祁门红茶加工过程中葡萄糖苷酶活性变化研究[J].安徽农业大学学报,2003,30(4):386-389.
[153]夏涛,童启庆,董尚胜,等.红茶萎凋发酵中葡萄糖苷酶的活性变化[J].茶叶科学,1996,16(1):63-66.
[154]赵东,刘祖生,奚彪.茶树多酚氧化酶基因的克隆及其序列比较[J].茶叶科学,21(2):94-98
[155]马春雷.茶树查尔酮异构酶、黄酮醇合成酶和无色花色素还原酶等基因的克隆与表达分析[D]中国农业科学院,硕士论文,2007-06-01.
[156]李远华,江昌俊,宛晓春.茶树咖啡碱合成酶基因mRNA表达的研究[J].茶叶科学，2004，21(1):23-28.
[157]Sugiyama T.,and Sadzuka Y.,1999,Combination of theaninewith doxorubicin inhibits hepatic metastasisof M5076ovar-ian sarcoma,Clinical Cancer Res.,5(2):413-416.
[158]陈暄,房婉萍,邹中伟,等.茶树冷胁迫诱导抗寒基因CBF的克隆与表达分析[J].茶叶科学2009,29(1):53-59.
[159]张亚丽.茶树ACS、ACO基因克隆与亲环素基因的鉴定及其表达分析[D].中国农业科学院,硕士论文,2007-06-01.
[160]刑浩然,刘丽娟,刘国振.植物蛋白质的亚细胞定位研究进展[J].华北农学报,2006,21:1-6.
[161]汤文文. BRSK2对能量压力诱导下细胞周期适应性变化的调控作用研究[D].上海:复旦大学,博士学位论文,2006-11-20.
[162]李远华,江昌俊,杨顺利,等.茶树葡萄糖苷酶cDNA克隆和原核表达[J].农业生物技术学报,2004,12(6):625-629.
[163]马振刚,唐婧,马淑华,等.新型碱性低分子量β-葡萄糖苷酶基因的分离与酶学性质研究[J].食品工业科技,2012,(4):108-113.
[164]唐婧.一株产纤维素酶细菌的分离及其β-葡萄糖苷酶基因克隆[D].西南大学,硕士学位论文,2008.
[165]张正竹.绿茶主要香气物质的糖昔类前体研究[D].湖南农业大学,博士学位论文,2000.
[166]李远华.茶树β-葡萄糖苷酶基因克隆、表达和分布[D].安徽农业大学,博士学位论文,2003-10.
[167] Haller F,Kulle B,Schwager S,et al,Equivalence test in quantitative reverse transcription polymerasechain reaction:confirmation of refernce genes suitable for normalization.Analytixal Biochemistry,2004,335:1-9.
[168]张珣.六种植物病毒Real Time PCR定量方法的建立及应用[D].北京:中国农业科学院.博士论文,2008.
[169]洪云,李津,汪和睦,等.实时荧光定量PCR技术进展[J].国际流行病学传染病学杂志,2006,33(3):161-166.
[170]高斌,刘敬忠.实时荧光PCR技术的研究及其应用进展[J].诊断学理论与实践,2005,4(6):507-509.