【作者】 孙云；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 茶学， 2009， 博士

【摘要】 抗坏血酸过氧化物酶(ascorbate peroxidase，APX)是植物活性氧代谢中重要的抗氧化酶之一，又是维生素C代谢的主要酶类。茶叶中富含维生素C，作为茶叶中重要活性营养成分，维生素C在茶叶加工和储存过程中易造成大量损失。开展茶叶中维生素C代谢相关基因的研究，将有助于了解茶叶中维生素C代谢的机制，为调控茶叶维生素C水平提供理论依据。本研究以茶叶(Camellia sinensis)为供试材料，研究了茶叶抗坏血酸过氧化物酶的生理特性及分子机理。通过对茶叶APX活性测定条件的探讨和建立，研究茶树不同品种、茶叶新梢不同生育时期、茶叶加工过程APX活性、同工酶的动态变化以及及维生素C含量的变化特点，探讨茶叶APX的代谢生理和作用机制；克隆了茶叶cAPX基因，并利用生物信息学方法对它的结构和功能进行预测和分析；在此基础上应用荧光定量PCR技术分析了茶叶cAPX基因的定量表达。研究结果如下：1．茶叶抗坏血酸过氧化物酶(APX)活性的测定方法建立探讨了茶树鲜叶抗坏血酸过氧化物酶(APX)活性的测定条件。结果表明，茶叶APX测定的最佳条件为，聚乙烯聚吡咯烷酮(PVPP)加入量约为鲜叶重的1．5倍，酶提取液pH为7．8，反应液pH为7．0，抗坏血酸(AsA)浓度为0．50 mmol／L时茶树鲜叶APX活性最高。试验样品APX活性随冷冻贮藏天数增加，不断降低，样品应可能保持芽叶的新鲜度。2．不同茶树品种、新梢不同生育时期APX酶活性研究选择福建代表性的绿茶品种(福鼎大白茶、福云6号)和乌龙茶品种(铁观音、毛蟹、黄旦、本山、肉桂)，分析了不同茶树品种以及茶树鲜叶不同叶位APX活性的变化。结果表明，福建主要7个茶树品种以福云6号的APX活性最高，铁观音的APX活性最低，绿茶品种的APX活性高于乌龙茶品种，不同品种APX活性大小依次为福云6号＞福鼎大白茶＞肉桂＞毛蟹＞黄旦＞本山＞铁观音；茶树鲜叶不同叶位APX活性变化趋势为：芽＞第一叶＞第二叶＞第三叶＞老叶＞嫩茎，随着鲜叶成熟度增加，APX活性下降。3．茶叶加工过程中APX活性及维生素C动态变化研究探讨茶叶不同萎凋程度及乌龙茶加工过程APX和维生素C的动态变化。结果表明，茶叶萎凋过程中APX活性呈“下降—上升—下降”趋势，相对活性由100％降至1．7％，萎凋不同阶段APX活性差异显著；萎凋过程中茶叶维生素C含量呈缓慢减少趋势，维生素C保留在鲜叶的80％以上，只经过萎凋的茶类(传统白茶)维生素C保留较为丰富。乌龙茶加工过程APX活性也呈“下降—上升—下降”趋势，相对活性由100％降至17．45％，炒青前各工序茶叶的APX活性差异显著，炒青后各工序茶叶的APX活性差异不显著；维生素C含量呈减少趋势，相对保留量由100％降至9．01％，揉捻工序维生素C含量下降幅度最大，其次是摇青工序。4．不同茶树品种及茶叶加工过程中APX同工酶的研究运用同工酶技术分析福建代表性绿茶、乌龙茶品种APX同工酶差异，分析探讨茶叶不同萎凋程度APX同工酶动态变化。结果表明，不同茶树品种APX同工酶存在差异，茶叶APX同工酶共有6条谱带，其中A2、A4、A5和A6四条为不同品种茶树所共有，A4、A5谱带为茶叶APX的重要同工酶。7个不同茶树品种的酶带，福鼎大白茶与毛蟹的酶带最亮最强，铁观音、黄旦与福云6号酶带最弱，除福云6号外，其他品种同工酶的表达量与APX活性存在相关性。茶叶萎凋茶叶APX同工酶谱带数量减少，起主要作用的为A4、A5同工酶也逐渐减弱，但萎凋18 h同工酶谱带强度与数量增加。同工酶的变化与APX酶活性具有相关性。5．茶叶APX的cDNA克隆从茶叶中克隆出了一个长度为1 083 bP的cAPX基因的cDNA序列，经测序分析，其包含750 bP的开放读码框，编码250个氨基酸。该基因在3’RACE与保守区重叠碱基数为321 bp，与5’RACE重叠碱基数是77 bp，非编码区5’有94 bp，3’有136 bp。已将该基因序列登录Genbank，登录号为Eu547804。6．茶叶APX基因的生物信息学分析利用生物信息学的方法对克隆的茶树cAPX基因的核酸序列及对应氨基酸序列的理化性质、结构特征、功能及系统演化关系等进行预测分析。结果表明，茶叶cAPX是一分子量27．53 kDa，等电点pI 5．59，比较稳定的酸性蛋白，属于亲水性蛋白，不含有跨膜信号，不具有信号肽；茶叶cAPX磷酸化位点有10个，丝氨酸(Ser)、酪氨酸(Tyr)磷酸化位点的各4个，苏氨酸(Thr)磷酸化位点2个，蛋白氨基酸序列9-31区域最有可能形成卷曲螺旋结构，α螺旋和不规则卷曲是茶叶cAPX蛋白最大量的结构元件。茶叶cAPX基因参与的生物过程主要是响应化学刺激和应激反应，具有四吡咯结合、过氧化物酶活性和氧化活性分子功能。蛋白的功能预测进一步证实茶叶cAPX属植物抗坏血酸过氧化物酶(Plant ascorbate-peroxidase)的蛋白质家族成员。7．茶叶主要品种及茶叶加工中APX基因的定量表达应用荧光定量PCR的方法对不同茶树品种APX基因的表达以及萎凋工艺过程中APX基因的表达进行定量分析。结果表明，茶树不同品种APX基因表达量存在显著差异，相对表达量变化范围为0．65-5．69，6个茶树品种APX基因相对表达量的大小为毛蟹＞福云6号＞肉桂＞福鼎大白茶＞黄旦＞铁观音。茶叶萎凋过程中APX基因的表达量呈现“升高—降低—再升高—再降低”的趋势，相对表达量变化范围为0．02-7．46，茶叶APX基因的表达明显受到萎凋工艺处理的影响。更多还原

【Abstract】 Ascorbate peroxidase (APX) is one of the key antioxidases in the process of activeoxygen metabolism in plants,furthermore,it is a major family of enzymes in the processof vitamin C (VC) metabolism.Tea contains abundant VC,which is an important activecomponent with abundant nutrition in tea.However,there will be a great loss of VC in teaduring processing and storage.Studies on related genes of VC metabolism in the tea plant(Camellai sinensis) will be helpful to uncover the mechanism of VC metabolism in tea,andwill provide theoretical basis for regulating VC level in tea.In this experiment,the tea leaves were used as the materials to study physiological andmolecular mechanism of APX in tea.After exploration and establishment test conditions oftea APX activity,changes in activity of APX were not only analyzed in different teacultivars but also during different growth periods of tea shoots.Moreover,the dynamicchanges of isoenzymes of APX were analysed both in different tea cultivars and during teaprocessing.Furthermore,the changes of VC contents were conducted during tea processing.cAPX gene was cloned from the leaf and the structure and functions were predicted andanalyzed by bioinformatics.On the basis of the studies above,the quantitative expressionof cAPX gene in tea was analyzed by fluorescence quantitative PCR technology.The mainresults showed as follows:1.Establishment of the test method of analysis of APX activity in teaThe test conditions of APX activity from tea fresh leaves were established in this study.And the optimal conditions of APX testing were as follows:amount ofpolyvinylpolypyrrolidone (PVPP) used was about 1.5 times the weight of fresh leaves;pHvalue of the enzyme extracting buffer was 7.8;pH value of reaction system was 7.0;thebest concentration of ascorbic acid (AsA) was 0.50 mmol/L.APX of high activity could beextracted by this method.It was found that APX activity of samples decreased continuouslywith the days of freezing storage prolonging,so the sample used should be as fresh aspossible.2.Analysis on APX activity both in different tea cultivars and during different growthperiods of tea shootsFujian representational green tea cultivars (FudingDabaicha,Fuyun 6) and oolong teacultivars(Tieguanyin,Maoxie,Huangdan,Benshan and Rougui) were selected to analyzethe changes of APX activity of different tea cultivars and tea fresh leaves from different positions.The results showed that APX activity of Funyun 6 was the highest among sevencultivars in Fujian and that of Tieguanyin was the lowest;APX activity of green teacultivars was higher than that of oolong tea cultivars;the ranking list of APX activity ofdifferent cultivars was as follows:Funyun 6＞Fudingdabaicha＞Rougui＞Maoxie＞Huangdan＞Benshan＞Tieguanyin.The ranking list of APX activity of tea fresh leaves indifferent positions was as shown below:the bud＞the first leaf＞the second leaf＞the thirdleaf＞the old leaf＞the young stemt,and the APX activity decreased along with the increaseof maturity of the fresh leaves.3.Analysis of the dynamic changes of APX activities and VC contents in tea during teaprocessingThe dynamic changes of the VC content and APX activity of leaves in differentwithering degrees and during oolong tea processing were explored.The results showed that:the change trend of APX activity during tea withering went up firstly,then down and upfinally,like a wave;the relative activity decreased from 100% to 1.7% during tea withering;the difference of APX activity at different withering phases was significant.Moreover,thecontent of VC decreased slowly during tea withering,and more than 80% of the VC contentwas not destroyed in the fresh leaf;the reserved quantity was more abundant in fresh leaf ofwhite tea,whose processing technique was only withering.The change trend of APXactivity during oolong tea processing was also“decrease-increase-decrease”,and therelative activity decreased from 100% to 17.45%.The difference of APX activity wassignificant in the techniques before chaoqing,while after was not,and the VC contentdecreased,and the relative contents decreased from 100% to 9.01%.The loss of VC contentwas the greatest in rolling technique,and yaoqing technique followed.4.Studies on APX isoenzymes both in different tea cultivars and during tea processingThe difference of APX isoenzymes of representational green and oolong tea cultivarsin Fujian was analyzed by isozyme technology,also the dynamic change of APXisoenzymes in different withering degrees was also explored.The results indicated thatthere were differences in expression of APX isoenzymes among different tea cultivars;and6 bands existed in the tea APX isoenzyme spectrum,among these,4 bands including A2,A4,A5 and A6 were in common in APX isoenzyme spectrum of different tea cultivars,andA4,A5 were more important isoenzymes in the tea plant.Enzyme belts of Fudingdabaichaand Maoxie were the brightest and strongest,and those of Tieguanyin,Huangdan andFunyun 6 were the weakest.Expression amount and activity of APX isoenzymes werecorrelative in different tea cultivars except the Fuyun 6 cultivar.Isoenzyme belts decreasedin the process of tea withering,and A4,A5 which played a major role were also weakengradually,but the strength and the amount ofAPX isoenzymes increased after withering for 18 hours.The changes of APX isoenzymes and its activity were correlative.5.cDNA cloning of tea APXThe full length of cDNA sequence(1083 bP) of cAPX gene from the tea plant wasobtained which contained 750 bp open reading frame,encoded 250 amino acids.Theoverlapped base of this cloned gene and the conserved region in 3′RACE and 5′RACE were 321 bp and 77 bp respectively.5′non-coding region were 94 bp,and 3′non-coding region were 136 bp.This gene was registered in GenBank,and the registeredNo.was EU547804.6.Analysis of bioinformatics of tea APX geneThe physicochemical properties,structure characteristics,functions and phylogeneticrelationship of tea APX gene nucleotide sequence and its amino acid sequences werepredicted and analyzed by bioinformatics in this experiment.The results showed that themolecular weight of cAPX was 27.53 kDa;and the theoretical isoelectric point (pI) was5.59.cAPX was a kind of steady and hydrophilic acidic protein without transmembranedomain and signal peptide.The phosphorylation sites of tea cAPX were 10,among these,the phosphorylation sites of both Set and Tyr were 4 and that of Thr were 2.The 9-31region of the protein amino acid sequences was the region most likely forming coiled-coilstructure.While the major structure components in the tea cAPX protein wereαspiral andirregular curl.The biological processes which tea APX gene participated in were mainly inresponse to chemical stimulation and stress response and antioxidant defense.Tea APXgene had the functions with tetrapyrrole combining,peroxidase activity and oxidizingbioactive molecule.Function prediction results further proved that tea cAPX proteinbelonged to the family of plant APX protein.7.Quantitative expression of APX gene in main tea cultivars and during teaprocessingExpression of APX gene in different tea cultivars and in the process of withering wasanalyzed by the fluorescence quantitative PCR technology.It was showed that theexpression amounts of APX gene in different tea cultivars were significantly different.Thechange range of relative expression amounts was from 0.65-5.69.The ranking list ofrelative expression amounts of APX gene in 6 tea cultivars was:Maoxie＞Fuyun 6＞Rougui＞Fudingdabaicha＞Huangdan＞Tieguanyin.During tea withering,the expressionamounts of APX gene increased at first,followed by a decline,and then increased again,finely decreased.And the change range of relative expression amounts was from 0.02 to7.46.At the same time,the expression of APX gene was affected by the treatment ofwithering technique.更多还原