茶多酚氧化酶基因的克隆及其工程菌的构建
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摘要
茶黄素作为一种茶叶所特有的色素物质，由于其具有极强的抗氧化及清除自由基的功能，近些年来受到研究者的广泛关注，并在保健和抗癌治癌等领域成为研究热点，是目前茶叶深度开发领域研究的热点成分，在天然药物、功能食品、日用化工、功能饮料等领域有着广泛的应用前景。茶黄素的合成机理是儿茶素在茶多酚氧化酶（PPO）的催化作用下氧化聚合形成邻醌，邻醌进一步缩合形成茶黄素。目前茶黄素的生产技术主要为体外酶促氧化的方法，其中多酚氧化酶是催化反应合成茶黄素的关键酶，如何得到大量、优质的酶源成为提高茶黄素生产技术的关键因素。
本文旨在通过基因工程的手段，利用融合蛋白表达系统，构建出茶PPO的基因表达载体，并进一步将其转入优势表达菌中，以期构建出茶PPO基因工程菌，并对其进行应用基础研究，从而解决茶黄素生产的关键酶源问题，进而为茶黄素的工业化生产提供一定的理论依据和研究基础。主要研究结果如下：
通过对各种CTAB法的综合与优化成功提取并纯化得到了纯度较高的迎霜茶树基因组DNA，并通过PCR扩增得到PPO的编码区片段。通过测序可知，迎霜茶树的PPO编码区序列长度为1800bp。并将其登录于GenBank，注册号为GQ214317。
对PPO的核酸序列以及翻译得到的氨基酸序列进行生物信息学分析。结果表明：所得到的迎霜品种茶树PPO基因与其他茶树基因都高度同源，相似性达到99%以上；PPO基因共编码599个氨基酸，分子式为C3008H4677N813O900S18，分子量为67207.2，理论等电点为6.24。PPO同属于酪氨酸酶、PPO1_DML和PPO1_KFDV三个超家族，且PPO的关键结构域Cu2+结合区具有高度的保守性；且PPO没有跨膜区，不存在信号肽，其存在于叶绿体的可能性最大，很有可能具有叶绿体转运肽。
将克隆得到的PPO基因重组到pET-28a表达载体上，构建出原核表达载体pET-PPO，并将其转入E. coli BL21（DE3），实现了PPO的原核诱导表达。原核表达过程中，目的蛋白容易形成包涵体，酶的活性较低；SDS-PAGE电泳检测结果表明，在71KD处有一条特异蛋白条带，与预测大小相符。
将克隆得到的PPO基因重组到pPICZα A融合蛋白表达载体上，构建出茶PPO的融合蛋白真核表达载体pPICZα-PPO2，并将其转入巴斯德毕赤酵母(Pichia pastoris)GS115中，成功筛选出多个阳性转化子。对阳性转化子进行甲醇诱导，采用Western-Blotting方法在培养液中成功检测到诱导表达的目的蛋白。酶活检测结果也显示诱导产物具有较高的相对酶活性，能够正确发挥酶蛋白的生物功能。
对PPO的真核蛋白表达进行优化策略研究，首先对PPO基因进行改造，构建出茶PPO的融合蛋白真核表达载体pPICZα-PPO3和pPICZα-PPO4。然后将pPICZα-PPO2、pPICZα-PPO3和pPICZα-PPO4分别转入巴斯德毕赤酵母GS115、KM71和SMD1168三种酵母菌种中，得到9种PPO的重组转化子，同时筛选出相应的阳性转化子，并对其进行甲醇诱导表达，Western-Blotting结果表明：9种组合中，只有转化了pPICZα-PPO2的GS115重组酵母得到了PPO阳性表达。
最后，对PPO基因工程菌进行应用基础研究显示：诱导表达的PPO的粗酶液酶活力为63.6U；遗传稳定性较好，在诱导3天后开始表达目的蛋白，并在7天后仍然保持目的蛋白的表达。
Theaflavin, as a peculiar pigment material of tea, since it has the function of strongresistance to oxidation and free radicals, in recent years, got a lot of attention by researchersand became a research hotspot in health care and cancer cure fields. Now it is the hotcomposition in the research of tea depth development fields, and has a widely Applicationprospect in the fields of natural medicines, functional foods, daily chemical products,functional drinks etc. Its synthesis mechanism is tea flavin catechins are oxidativepolymerized into the o-quinones under the action of tea polyphenol oxidase (PPO), then theo-quinones occur a condensation reaction into theaflavins. At present the main productiontechnology of theaflavins is vitro enzyme oxidation method, thereinto polyphenol oxidase isthe key enzyme for catalytic oxidation forming into theaflavins. How to get a lot of highquality enzyme source is the key factoe to improve the production technology of theaflavins.
This paper aims at constructing the expression vector of tea PPO, through geneticengineering means, with the fusion protein expression system, and further to transform it intothe advantage expression strains, in order to construct the tea PPO’s genetic engineering strain,and its applied foundation research was studyed, so as to solve the key enzyme sourceproblem of the production of theaflavins, then to provide the theoretical basis and basicresearch for the industrial production of theaflavins. The main research results are as follows:
Through synthesizing and optimizing the CTAB method, the highly purfied genomicDNA of Yingshuang tea plant was extracted and purified. Then the PPO’s cds sequence wasamplified by PCR from the genome with the high fidelity polymerase LA-taq. The sequencingresults showed that the cds sequence’s length of PPO was1800bp. The sequence had beensigned in the Genbank and its registered number is GQ214317.
The bioinformatics analysis result showed that the Yingshuang tea PPO gene was highlyhomologous with the other tea PPO genes, the similarity was above99%; Its gene encoding atotal599amino acids, and its molecular formula was C3008H4677N813O900S18, molecular weightwas67207.2, the theoretical isoelectric point was6.24; PPO belongs to three superfamily(Tyrosinase. PPO1_DML and PPO1_KFDV), and the key Cu2+combined structure domain of PPO was highly conservative; It has no transmembrane area and no signal peptide,and most likely exists in the chloroplast with the chloroplasts transport peptides.
The cloned PPO1gene was recombined to the pET-28vector to construct the expressionvector pET-PPO1, and transform it into the E.coli BL21(DE3). Then the prokaryoticexpression of PPO was successfully realized by being induced with IPTG. During theprokaryotic expression process, PPO easily formed to the inclusion body, and the enzymeactivity was lower; A71KD protein was screened by SDS-PAGE electrophoretic and the sizewas same with the prediction.
The cloned PPO2gene was recombined to the pPICZα vector to construct the expressionvector pPICZα-PPO2, and transform it into the Pichia pastoris GS115. Many positivetransformants were successfully screened by PCR, and were induced by methanol. Thepurpose protein was successfully detected in the culture medium by using Western-Blottingmethod. Enzyme activity detection result showed that the induction product had high relativeenzyme activity, and could correctly play the biological function.
The optimized strategy research of PPO’s eukaryotic expression was made as follows:Firstly we modificated the gene of PPO, and constructed the fusion protein expression vectorof PPO (pPICZα-PPO3and pPICZα-PPO4), then transformed them (include pPICZα-PPO2)into the Pichia pastoris GS115, KM71and SMD1168to get9kinds of PPO recombinedtransformants. The corresponding positive transformants were screened by PCR, and wereinduced by methanol. Western-Blotting results show that: in the nine combinations, only thepPICZα-PPO2/GS115got PPO positive expression.
Finally, the basic applied research of PPO genetic engineering strain showed that: theenzymatic activity of the crude enzyme liquid was63.6U; The genetic stability is good, thetarget protein was in expressed after3days induced, and still kept expression of the targetprotein in7days.
本文旨在通过基因工程的手段，利用融合蛋白表达系统，构建出茶PPO的基因表达载体，并进一步将其转入优势表达菌中，以期构建出茶PPO基因工程菌，并对其进行应用基础研究，从而解决茶黄素生产的关键酶源问题，进而为茶黄素的工业化生产提供一定的理论依据和研究基础。主要研究结果如下：
通过对各种CTAB法的综合与优化成功提取并纯化得到了纯度较高的迎霜茶树基因组DNA，并通过PCR扩增得到PPO的编码区片段。通过测序可知，迎霜茶树的PPO编码区序列长度为1800bp。并将其登录于GenBank，注册号为GQ214317。
对PPO的核酸序列以及翻译得到的氨基酸序列进行生物信息学分析。结果表明：所得到的迎霜品种茶树PPO基因与其他茶树基因都高度同源，相似性达到99%以上；PPO基因共编码599个氨基酸，分子式为C3008H4677N813O900S18，分子量为67207.2，理论等电点为6.24。PPO同属于酪氨酸酶、PPO1_DML和PPO1_KFDV三个超家族，且PPO的关键结构域Cu2+结合区具有高度的保守性；且PPO没有跨膜区，不存在信号肽，其存在于叶绿体的可能性最大，很有可能具有叶绿体转运肽。
将克隆得到的PPO基因重组到pET-28a表达载体上，构建出原核表达载体pET-PPO，并将其转入E. coli BL21（DE3），实现了PPO的原核诱导表达。原核表达过程中，目的蛋白容易形成包涵体，酶的活性较低；SDS-PAGE电泳检测结果表明，在71KD处有一条特异蛋白条带，与预测大小相符。
将克隆得到的PPO基因重组到pPICZα A融合蛋白表达载体上，构建出茶PPO的融合蛋白真核表达载体pPICZα-PPO2，并将其转入巴斯德毕赤酵母(Pichia pastoris)GS115中，成功筛选出多个阳性转化子。对阳性转化子进行甲醇诱导，采用Western-Blotting方法在培养液中成功检测到诱导表达的目的蛋白。酶活检测结果也显示诱导产物具有较高的相对酶活性，能够正确发挥酶蛋白的生物功能。
对PPO的真核蛋白表达进行优化策略研究，首先对PPO基因进行改造，构建出茶PPO的融合蛋白真核表达载体pPICZα-PPO3和pPICZα-PPO4。然后将pPICZα-PPO2、pPICZα-PPO3和pPICZα-PPO4分别转入巴斯德毕赤酵母GS115、KM71和SMD1168三种酵母菌种中，得到9种PPO的重组转化子，同时筛选出相应的阳性转化子，并对其进行甲醇诱导表达，Western-Blotting结果表明：9种组合中，只有转化了pPICZα-PPO2的GS115重组酵母得到了PPO阳性表达。
最后，对PPO基因工程菌进行应用基础研究显示：诱导表达的PPO的粗酶液酶活力为63.6U；遗传稳定性较好，在诱导3天后开始表达目的蛋白，并在7天后仍然保持目的蛋白的表达。
Theaflavin, as a peculiar pigment material of tea, since it has the function of strongresistance to oxidation and free radicals, in recent years, got a lot of attention by researchersand became a research hotspot in health care and cancer cure fields. Now it is the hotcomposition in the research of tea depth development fields, and has a widely Applicationprospect in the fields of natural medicines, functional foods, daily chemical products,functional drinks etc. Its synthesis mechanism is tea flavin catechins are oxidativepolymerized into the o-quinones under the action of tea polyphenol oxidase (PPO), then theo-quinones occur a condensation reaction into theaflavins. At present the main productiontechnology of theaflavins is vitro enzyme oxidation method, thereinto polyphenol oxidase isthe key enzyme for catalytic oxidation forming into theaflavins. How to get a lot of highquality enzyme source is the key factoe to improve the production technology of theaflavins.
This paper aims at constructing the expression vector of tea PPO, through geneticengineering means, with the fusion protein expression system, and further to transform it intothe advantage expression strains, in order to construct the tea PPO’s genetic engineering strain,and its applied foundation research was studyed, so as to solve the key enzyme sourceproblem of the production of theaflavins, then to provide the theoretical basis and basicresearch for the industrial production of theaflavins. The main research results are as follows:
Through synthesizing and optimizing the CTAB method, the highly purfied genomicDNA of Yingshuang tea plant was extracted and purified. Then the PPO’s cds sequence wasamplified by PCR from the genome with the high fidelity polymerase LA-taq. The sequencingresults showed that the cds sequence’s length of PPO was1800bp. The sequence had beensigned in the Genbank and its registered number is GQ214317.
The bioinformatics analysis result showed that the Yingshuang tea PPO gene was highlyhomologous with the other tea PPO genes, the similarity was above99%; Its gene encoding atotal599amino acids, and its molecular formula was C3008H4677N813O900S18, molecular weightwas67207.2, the theoretical isoelectric point was6.24; PPO belongs to three superfamily(Tyrosinase. PPO1_DML and PPO1_KFDV), and the key Cu2+combined structure domain of PPO was highly conservative; It has no transmembrane area and no signal peptide,and most likely exists in the chloroplast with the chloroplasts transport peptides.
The cloned PPO1gene was recombined to the pET-28vector to construct the expressionvector pET-PPO1, and transform it into the E.coli BL21(DE3). Then the prokaryoticexpression of PPO was successfully realized by being induced with IPTG. During theprokaryotic expression process, PPO easily formed to the inclusion body, and the enzymeactivity was lower; A71KD protein was screened by SDS-PAGE electrophoretic and the sizewas same with the prediction.
The cloned PPO2gene was recombined to the pPICZα vector to construct the expressionvector pPICZα-PPO2, and transform it into the Pichia pastoris GS115. Many positivetransformants were successfully screened by PCR, and were induced by methanol. Thepurpose protein was successfully detected in the culture medium by using Western-Blottingmethod. Enzyme activity detection result showed that the induction product had high relativeenzyme activity, and could correctly play the biological function.
The optimized strategy research of PPO’s eukaryotic expression was made as follows:Firstly we modificated the gene of PPO, and constructed the fusion protein expression vectorof PPO (pPICZα-PPO3and pPICZα-PPO4), then transformed them (include pPICZα-PPO2)into the Pichia pastoris GS115, KM71and SMD1168to get9kinds of PPO recombinedtransformants. The corresponding positive transformants were screened by PCR, and wereinduced by methanol. Western-Blotting results show that: in the nine combinations, only thepPICZα-PPO2/GS115got PPO positive expression.
Finally, the basic applied research of PPO genetic engineering strain showed that: theenzymatic activity of the crude enzyme liquid was63.6U; The genetic stability is good, thetarget protein was in expressed after3days induced, and still kept expression of the targetprotein in7days.
引文
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