茶树体内茶氨酸合成酶的克隆与异源表达及一氧化氮信号对其调控的研究
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摘要
茶是目前世界上饮用量最大的非酒精类饮料，L-茶氨酸是茶树体内特征性非蛋白质氨基酸，不仅决定了茶的风味还有非常广泛的药理作用。茶氨酸合成酶（Theanine synthetase，TS）不仅是茶氨酸合成的关键酶，也对茶树体内的氮代谢有重大影响。研究表明，茶氨酸合成酶基因序列与谷氨酰胺合成酶（Glutaminesynthetase, GS）基因序列有高度同源性，有可能是谷氨酰胺合成酶基因家族成员，对其进行研究，不仅有利于掌握茶氨酸代谢与调控的规律，也有助于了解茶树体内氮同化与转运的机理。本研究利用RT-PCR的方法克隆了茶树体内茶氨酸合成酶基因（DD410895）与谷氨酰胺合成酶基因（AB115184），通过原核与真核表达进行功能验证，并将原核表达的蛋白纯化以制备多抗；在此基础上，利用生物信息学的手段对TS与GS推导出的蛋白质序列进行结构与功能的分析，并利用系统进化树的方法对它们在GS基因家族中的地位进行聚类分析；利用HPLC检测与Western Blotting相结合的方法对不同NO诱导处理下茶籽苗中TS的表达与茶氨酸的积累进行分析。通过上述研究得到以下结果：
通过提取茶树根部与叶部总RNA，进行RT-PCR，克隆得到茶树茶氨酸合成酶基因（DD410895）与谷氨酰胺合成酶基因（AB115184）。获得的TS基因长度为1071bp，与已知的茶树TS基因的氨基酸序列有3个氨基酸的差异，与其他植物GS基因具有较高的同源性，获得的GS基因与已知的茶树GS基因的氨基酸序列有1个氨基酸的差异。对两条基因进行原核表达后得到可溶性蛋白，进行酶活力验证，结果表明，表达的TS蛋白能够催化谷氨酰胺与盐酸乙胺合成茶氨酸，而不能催化谷氨酸与盐酸乙胺合成茶氨酸，也不具备GS的催化活力，酶促反应产生的产物用HPLC与ESI-MS进行鉴定，确定为茶氨酸；而表达的GS蛋白明显具有转氨基的催化活力，相较于细菌本身GS催化能力大大增强。
构建植物双元表达载体pGREEN-35S-TS/GS，导入农杆菌，运用花序侵染法转入拟南芥，分别获得转茶TS和GS基因的转基因拟南芥，通过除草剂Basta对后代进行筛选，并利用PCR的方法对阳性植株进行鉴定。利用载体上的GFP标签对转TS基因的拟南芥根尖细胞进行激光共聚焦显微镜观察，发现融合蛋白主要存在于细胞质中。
用Expasy软件包和CBS，Swiss-Model，InterProScan等网站的在线分析工具对TS与GS的理化特性，蛋白结构等进行分析与预测。结果表明TS的理论等电点PI=5.52，蛋白质相对分子质量MW=39306.3Da，GS的理论等电点PI=6.13，蛋白质相对分子质量MW=39240.3Da，二者均为易溶、亲水性强的蛋白，不存在跨膜结构。二级结构预测表明二者都是混合型蛋白结构差异不大。氨基酸序列和结构分析显示TS与GS蛋白均包含了一个GS beta-Grasp结构域和一个GS催化结构域，这些功能区在植物的谷氨酰胺合成酶中是保守的，均为Gln-synt结构域。运用软件对蛋白的细胞定位和功能分析表明，TS与GS蛋白主要定位于细胞质内，具有转录、转录调控和信号转导功能的概率较高。运用同源建模软件SWISS-PdbView对TS三维结构进行预测，并以玉米GS蛋白三维结构为模板（编号:2d3a）用metapocket软件对TS蛋白活性中心进行分析，预测出三个酶反应的结合位点，其位置与GS大致相当，但是其所能结合的氨基酸位点有着细微的差别，可能就是二者催化反应差异的原因所在。运用Mega4.1软件构建了GS的系统进化树，表明TS属于GS家族成员，并且可以将山茶属GS蛋白划分为3类。
利用外源一氧化氮（NO）供体硝普钠（SNP）提供不同浓度的NO处理幼嫩茶籽苗，发现能够明显促进TS的表达和茶氨酸的积累，短期内NO浓度的提高能明显增强茶籽苗体内茶氨酸的含量，但是达到一定的临界值后这种作用不再呈正相关，而处理一段时间后，NO的刺激作用明显被茶籽苗自身生长发育过程所调节，茶氨酸的积累量逐步下降，使用NO清除剂与NOS抑制剂能明显阻断外源NO的刺激作用，但不会影响茶籽苗茶氨酸的合成与代谢的正常通路。说明适当剂量的外源NO可以直接影响TS基因的表达，但尚不明确这种作用是直接作用于蛋白结构上还是与其他信号级联系统共同作用的结果。NO的信号调节作用还对EGCG的合成有明显的关联，不过对其他游离氨基酸与儿茶素代谢的影响并不明显。
Tea is the most consumed non-alcoholic beverages in the world. L-theanine is thecharacteristic and non-protein amino acid in tea plants (Camellia sinensis), which notonly determines the flavor of tea but also has a wide range of pharmacologicalfunctions. Theanine synthetase is a key enzyme in the theanine synthesis, and plays akey role in nitrogen metabolism in tea plants. Previous repots showed that theaninesynthetase gene sequence is highly homologous with glutamine synthetase genesequences. Further studies are needed for our better understanding theaninemetabolism and the mechanism of nitrogen assimilation and transport in tea plants. Inthis study, we cloned theanine synthetase gene (DD410895) and glutamine synthetasegene (AB115184) from tea by RT-PCR, and verified its function through theprokaryotic and eukaryotic expression, and prepared polyclonal antibody throughpurification prokaryotic expression protein. On this basis, we used bioinformaticstools to analyze the structure and function of these proteins, which deduced by TS andGS gene sequences, and using the method of phylogenetic tree to analysis theirlocalization in the GS gene family. Moreover, we analyzed the expression of TS geneand theanine accumulation in tea seedling treated with different NO concentrations,by using the methods of HPLC and Western Blotting techology，respectively. Hereare the detailed results:
Total RNA of tea roots and leaves were extracted, then theanine synthetase gene(DD410895) and glutamine synthetase gene (AB115184) were cloned by RT-PCR.The length of obtained TS gene was1071bp, which had three amino acid residuesdifferent from the known amino acid sequences of TS gene in tea, and high homologywith GS genes in plants. The obtained GS gene had the only one amino acid differentfrom the known amino acid sequences of GS gene. The two genes were expression inE.coli, and soluble proteins were obtained. Specificities of the enzyme function werefurther verified. These results show that the expressed protein of TS catalyzestheanine synthesis using glutamine and ethylamine hydrochloride, rather thanglutamic acid and ethylamine hydrochloride. Tea TS has no catalytic activity of GS.Theanine，the product of enzymatic reaction, was identified by HPLC and ESI-MS.GS protein has a catalytic activity for amino transportation, which is much greater than the activity of bacteria GS.
Plant binary expression vectors pGREEN-35S-TS/GS, containing TS or GS cDNAsfused with GFP, were constructed and introduced into Agrobacterium, and furthertransferred into the Arabidopsis by the floral dip method. The transgenic Arabidopsiscontaining TS and GS genes was screened by herbicide Basta. The positive plantswere confirmed by PCR. The fusion protein was observed mainly in the cytoplasmin the root tip cells of the transgenic Arabidopsis by using laser scanning confocalmicroscope.
The Expasy software packages and some online analysis tools such as CBS,Swiss-Model and InterProScan were used to analyse and predict the physical andchemical properties and the protein structure of TS and GS gene. The results showthat TS theoretical isoelectric point (PI) was5.52, protein molecular weight (MW) is39306.3Da, GS theoretical isoelectric point (PI) is6.13, and protein molecular weight(MW) is39240.3Da. Both of them are soluble, strong hydrophilic proteins, whichhave no transmembrane structure. Secondary structure prediction indicates that theyare all mixed type proteins and little difference between the protein structures. Aminoacid sequence and structural analysis showed that both TS and GS protein includes aGS beta-Grasp domain and a GS catalytic domain, these functional fragments areconserved in glutamine synthetase genes of many plant species and all belong toGln-synt domains. Protein subcellular localization and functional analyses showedthat, TS and GS proteins were localized in the cytoplasm, which have high probabilityto executive some function such as transcription, transcription regulation and signaltransduction. Homology modeling software SWISS-PdbView was used to predictthree-dimensional structure of the TS gene. Moreover, the metapocket software wasused to analyze TS protein active sites through three-dimensional structure of maizeGS protein as a template (No.:2d3a). Three binding sites in the enzyme werepredicted, roughly equivalent to its location with GS, but its sites which couldcombinate amino acid have a slight difference, which is probably the reason for thedifference of the catalytic reaction between the TS and GS. Using Mega4.1software,the phylogenetic tree was built, which indicates that the TS gene belonging to GSfamily members and GS protein of Camellia sinensis can be classified into threecategories.
High-performance liquid chromatography (HPLC) detector results revealed that nitrogen monoxide (NO) induced the rapid accumulation of L-theanine in the tea rootrather than in the leaves, and the expression of L-theanine synthetase was bothenhanced in both the roots and leaves. HPLC results also revealed the presence of NOinduced the accumulation of catechins as ester-like compounds in the leaves.Application of NO scavengers and pretreatment with NO-metabolism inhibitorsreduced NO-induced L-theanine accumulation, but no significant effect on catechinsynthesis. These results indicated that L-theanine and catechin metabolism areregulated by different mechanisms in young tea seedlings.
通过提取茶树根部与叶部总RNA，进行RT-PCR，克隆得到茶树茶氨酸合成酶基因（DD410895）与谷氨酰胺合成酶基因（AB115184）。获得的TS基因长度为1071bp，与已知的茶树TS基因的氨基酸序列有3个氨基酸的差异，与其他植物GS基因具有较高的同源性，获得的GS基因与已知的茶树GS基因的氨基酸序列有1个氨基酸的差异。对两条基因进行原核表达后得到可溶性蛋白，进行酶活力验证，结果表明，表达的TS蛋白能够催化谷氨酰胺与盐酸乙胺合成茶氨酸，而不能催化谷氨酸与盐酸乙胺合成茶氨酸，也不具备GS的催化活力，酶促反应产生的产物用HPLC与ESI-MS进行鉴定，确定为茶氨酸；而表达的GS蛋白明显具有转氨基的催化活力，相较于细菌本身GS催化能力大大增强。
构建植物双元表达载体pGREEN-35S-TS/GS，导入农杆菌，运用花序侵染法转入拟南芥，分别获得转茶TS和GS基因的转基因拟南芥，通过除草剂Basta对后代进行筛选，并利用PCR的方法对阳性植株进行鉴定。利用载体上的GFP标签对转TS基因的拟南芥根尖细胞进行激光共聚焦显微镜观察，发现融合蛋白主要存在于细胞质中。
用Expasy软件包和CBS，Swiss-Model，InterProScan等网站的在线分析工具对TS与GS的理化特性，蛋白结构等进行分析与预测。结果表明TS的理论等电点PI=5.52，蛋白质相对分子质量MW=39306.3Da，GS的理论等电点PI=6.13，蛋白质相对分子质量MW=39240.3Da，二者均为易溶、亲水性强的蛋白，不存在跨膜结构。二级结构预测表明二者都是混合型蛋白结构差异不大。氨基酸序列和结构分析显示TS与GS蛋白均包含了一个GS beta-Grasp结构域和一个GS催化结构域，这些功能区在植物的谷氨酰胺合成酶中是保守的，均为Gln-synt结构域。运用软件对蛋白的细胞定位和功能分析表明，TS与GS蛋白主要定位于细胞质内，具有转录、转录调控和信号转导功能的概率较高。运用同源建模软件SWISS-PdbView对TS三维结构进行预测，并以玉米GS蛋白三维结构为模板（编号:2d3a）用metapocket软件对TS蛋白活性中心进行分析，预测出三个酶反应的结合位点，其位置与GS大致相当，但是其所能结合的氨基酸位点有着细微的差别，可能就是二者催化反应差异的原因所在。运用Mega4.1软件构建了GS的系统进化树，表明TS属于GS家族成员，并且可以将山茶属GS蛋白划分为3类。
利用外源一氧化氮（NO）供体硝普钠（SNP）提供不同浓度的NO处理幼嫩茶籽苗，发现能够明显促进TS的表达和茶氨酸的积累，短期内NO浓度的提高能明显增强茶籽苗体内茶氨酸的含量，但是达到一定的临界值后这种作用不再呈正相关，而处理一段时间后，NO的刺激作用明显被茶籽苗自身生长发育过程所调节，茶氨酸的积累量逐步下降，使用NO清除剂与NOS抑制剂能明显阻断外源NO的刺激作用，但不会影响茶籽苗茶氨酸的合成与代谢的正常通路。说明适当剂量的外源NO可以直接影响TS基因的表达，但尚不明确这种作用是直接作用于蛋白结构上还是与其他信号级联系统共同作用的结果。NO的信号调节作用还对EGCG的合成有明显的关联，不过对其他游离氨基酸与儿茶素代谢的影响并不明显。
Tea is the most consumed non-alcoholic beverages in the world. L-theanine is thecharacteristic and non-protein amino acid in tea plants (Camellia sinensis), which notonly determines the flavor of tea but also has a wide range of pharmacologicalfunctions. Theanine synthetase is a key enzyme in the theanine synthesis, and plays akey role in nitrogen metabolism in tea plants. Previous repots showed that theaninesynthetase gene sequence is highly homologous with glutamine synthetase genesequences. Further studies are needed for our better understanding theaninemetabolism and the mechanism of nitrogen assimilation and transport in tea plants. Inthis study, we cloned theanine synthetase gene (DD410895) and glutamine synthetasegene (AB115184) from tea by RT-PCR, and verified its function through theprokaryotic and eukaryotic expression, and prepared polyclonal antibody throughpurification prokaryotic expression protein. On this basis, we used bioinformaticstools to analyze the structure and function of these proteins, which deduced by TS andGS gene sequences, and using the method of phylogenetic tree to analysis theirlocalization in the GS gene family. Moreover, we analyzed the expression of TS geneand theanine accumulation in tea seedling treated with different NO concentrations,by using the methods of HPLC and Western Blotting techology，respectively. Hereare the detailed results:
Total RNA of tea roots and leaves were extracted, then theanine synthetase gene(DD410895) and glutamine synthetase gene (AB115184) were cloned by RT-PCR.The length of obtained TS gene was1071bp, which had three amino acid residuesdifferent from the known amino acid sequences of TS gene in tea, and high homologywith GS genes in plants. The obtained GS gene had the only one amino acid differentfrom the known amino acid sequences of GS gene. The two genes were expression inE.coli, and soluble proteins were obtained. Specificities of the enzyme function werefurther verified. These results show that the expressed protein of TS catalyzestheanine synthesis using glutamine and ethylamine hydrochloride, rather thanglutamic acid and ethylamine hydrochloride. Tea TS has no catalytic activity of GS.Theanine，the product of enzymatic reaction, was identified by HPLC and ESI-MS.GS protein has a catalytic activity for amino transportation, which is much greater than the activity of bacteria GS.
Plant binary expression vectors pGREEN-35S-TS/GS, containing TS or GS cDNAsfused with GFP, were constructed and introduced into Agrobacterium, and furthertransferred into the Arabidopsis by the floral dip method. The transgenic Arabidopsiscontaining TS and GS genes was screened by herbicide Basta. The positive plantswere confirmed by PCR. The fusion protein was observed mainly in the cytoplasmin the root tip cells of the transgenic Arabidopsis by using laser scanning confocalmicroscope.
The Expasy software packages and some online analysis tools such as CBS,Swiss-Model and InterProScan were used to analyse and predict the physical andchemical properties and the protein structure of TS and GS gene. The results showthat TS theoretical isoelectric point (PI) was5.52, protein molecular weight (MW) is39306.3Da, GS theoretical isoelectric point (PI) is6.13, and protein molecular weight(MW) is39240.3Da. Both of them are soluble, strong hydrophilic proteins, whichhave no transmembrane structure. Secondary structure prediction indicates that theyare all mixed type proteins and little difference between the protein structures. Aminoacid sequence and structural analysis showed that both TS and GS protein includes aGS beta-Grasp domain and a GS catalytic domain, these functional fragments areconserved in glutamine synthetase genes of many plant species and all belong toGln-synt domains. Protein subcellular localization and functional analyses showedthat, TS and GS proteins were localized in the cytoplasm, which have high probabilityto executive some function such as transcription, transcription regulation and signaltransduction. Homology modeling software SWISS-PdbView was used to predictthree-dimensional structure of the TS gene. Moreover, the metapocket software wasused to analyze TS protein active sites through three-dimensional structure of maizeGS protein as a template (No.:2d3a). Three binding sites in the enzyme werepredicted, roughly equivalent to its location with GS, but its sites which couldcombinate amino acid have a slight difference, which is probably the reason for thedifference of the catalytic reaction between the TS and GS. Using Mega4.1software,the phylogenetic tree was built, which indicates that the TS gene belonging to GSfamily members and GS protein of Camellia sinensis can be classified into threecategories.
High-performance liquid chromatography (HPLC) detector results revealed that nitrogen monoxide (NO) induced the rapid accumulation of L-theanine in the tea rootrather than in the leaves, and the expression of L-theanine synthetase was bothenhanced in both the roots and leaves. HPLC results also revealed the presence of NOinduced the accumulation of catechins as ester-like compounds in the leaves.Application of NO scavengers and pretreatment with NO-metabolism inhibitorsreduced NO-induced L-theanine accumulation, but no significant effect on catechinsynthesis. These results indicated that L-theanine and catechin metabolism areregulated by different mechanisms in young tea seedlings.
引文
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