Studies On Physiology And Molecular Biology Of Tea (Camellia Sinensis) Ascorbate Peroxidase (APX)

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.