安吉白茶高氨基酸性状相关基因的全长cDNA克隆及功能的初步研究
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摘要
茶树(Camellia sinensis(L.) O. Kuntze)是我国重要的经济作物之一,为多年生常绿木本植物,其作为栽培作物在中国已有3000多年的历史。安吉白茶是茶树中的一个特异品种,其特异性主要表现在春季新梢叶色的可逆性白化现象,其在白化过程中伴随着新梢叶绿素含量的急剧下降与氨基酸含量的显著上升,氨基酸含量是普通茶树品种的2倍左右。茶氨酸(L-Theanine)是茶树的特征性成分,是一种非蛋白质氨基酸,占茶树氨基酸总量的50%-60%,为茶树中具有重要生理活性的次生代谢产物之一。前期研究以安吉白茶为材料,采用SSH、cDNA-AFLP等技术已筛选出一批可能与安吉白茶高氨基酸性状存在直接或间接关联的差异表达基因。本研究在此基础上仍以具有高氨基酸含量的珍稀茶树资源--安吉白茶为材料,克隆与安吉白茶高氨基酸性状相关的基因,并对其功能进行初步分析,旨在为调控茶树氨基酸生物合成及提高茶树氨基酸水平提供理论与技术支撑。本研究的主要结果如下：
1.采用Waters公司AccQ.Tag柱前衍生高效液相色谱法对安吉白茶全白期与全绿期叶片中的氨基酸组分及含量进行检测,结果表明,安吉白茶的全白期与全绿期叶都含有包括茶氨酸在内的18种氨基酸,但两个时期氨基酸各组分的含量存在较大差异,安吉白茶全白期一芽二叶中的氨基酸含量达到5.93%,是全绿期一芽二叶中的2.12倍。且相同时期一芽二叶中的氨基酸含量高于第三、四叶中的氨基酸含量。茶氨酸含量与氨基酸总量呈正相关,安吉白茶全白期叶中的茶氨酸含量为3.33%,是全绿期叶中茶氨酸含量的2.31倍。本研究还表明AccQ.Tag柱前衍生高效液相色谱法能较好的分离茶树中的各氨基酸组分,有利于对不同样品进行分析与比较。
2.以安吉白茶全白期与全绿期叶为研究材料,采用SSH技术,成功构建了安吉白茶全白期与全绿期叶片的抑制消减杂交cDNA文库,筛选出了一批差异表达基因,并对相关差异表达基因进行了初步分析,发现丝氨酸-乙醛酸转氨酶基因,丝氨酸羟甲基转移酶基因可能与安吉白茶高氨基酸性状相关,因而有进一步研究的价值,其后续研究正在进行中,可望为阐明安吉白茶全白期氨基酸超常富集的分子机理提供重要信息。
3.茶氨酸合成酶在反应底物L-谷氨酸和乙胺的存在下,由ATP提供能量,能催化合成茶氨酸。本试验利用SMART RACE技术首次成功克隆了安吉白茶茶氨酸合成酶基因(TS)的全长cDNA序列。TS基因的cDNA全长为1503bp, ORF为1071 bp,编码356个氨基酸。经相关生物信息学分析,TS的氨基酸残基数为356,分子量为39250.3(Da),理论等电点(PI)为5.79,属于亲水性蛋白,TS蛋白不存在信号肽序列,属于非分泌蛋白,非跨膜蛋白,其磷酸化位点有26个。其氨基酸序列中可能不存在卷曲螺旋结构。通过亚细胞定位预测,安吉白茶TS蛋白是一种细胞质蛋白。克隆茶树茶氨酸合成酶基因的全长cDNA,对于利用生物技术手段改良茶树品种、以调控提高茶氨酸含量具有重要意义。
4.采用原核表达和体外酶试验法,对茶氨酸合成酶基因的功能进行了初步验证。通过中间质粒pMD 19-T-TS,成功构建了茶树TS蛋白的重组质粒pET-32a(+)-TS,并转入表达载体BL21,经IPTG诱导实现了重组蛋白的表达,且表达蛋白的分子量与预测的一致,约为59 kD。采用L-谷氨酸和乙胺为底物,利用重组蛋白进行体外酶催化试验,经HPLC检测表明,重组蛋白成功催化了茶氨酸的合成。
5.利用SMART RACE技术首次成功克隆了安吉白茶纺锤体分解相关蛋白基因(CDC48)的全长cDNA序列。其cDNA全长为2859bp, ORF为2424 bp,共编码807个氨基酸。经相关生物信息学分析表明,CDC48的氨基酸残基数为807,分子量为89901.9(Da),理论等电点(PI)为5.16,属于亲水性蛋白,CDC48不存在信号肽序列,属于非分泌蛋白,非跨膜蛋白,不发生跨膜运动,磷酸化位点有38个,它们不均匀分布于整个多肽链中,其氨基酸序列有三个区域可能形成卷曲螺旋。通过亚细胞定位预测,认为安吉白茶CDC48蛋白是一种细胞核蛋白,主要定位在核区。CDC48是参与细胞周期调控的重要元素,研究表明,CDC48与泛素依赖蛋白降解途径有关,所以克隆CDC48基因的全长cDNA,研究其蛋白质的结构和功能,对于探明安吉白茶阶段性返白过程中氨基酸超常富集的分子机理具有重要意义。
6.采用相对定量qPCR技术,以茶树18s rRNA为内参基因,以安吉白茶全绿期叶为对照组,对安吉白茶全白期TS基因和CDC48基因的相对表达水平进行了定量分析,结果表明TS基因和CDC48基因在安吉白茶白期叶中的表达水平比绿期叶的。TS催化L-谷氨酸和乙胺生成茶氨酸；而CDC48与泛素依赖蛋白降解途径有关,其在安吉白茶全白期叶中表达水平较高,可能与蛋白质降解,氨基酸富集相关。因此,这两个基因在安吉白茶全白期叶中表达水平高于绿期叶,可能与该时期氨基酸含量较高的性状相关。
Tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important economy crops. It is an evergreen bush and has over 3000 year history as a cultivation crop in China. Anji Baicha is a kind of specific variety with reversible albinism in spring shoots. During the albescent stage, the content of chlorophyll in albino leaves sharply reduces, but the amount of amino acids raise significantly. The amount of amino acids is about 2 times higher in albino leaves than that of normal leaves from ordinary varieties. L-Theanine is a characteristic ingredient of tea plant, and a non-protein amino acid, which accounts for 50～60% of total amount of amino acids in tea plant. L-Theanine is a kind of secondary metabolite with important physiology activities. Our previous research had isolated a bunch of differentially expressed genes related to the property of high amino acid level during stage albescent process of Anji Baicha by SSH and cDNA-AFLP. In this study, the full-length cDNA of two genes were cloned and their functions were further analyzed. The research results would support to regulate enhancing of the L-theanine level in tea plant from both theoretical and practical aspects. The main results were as follows:
The components and contents of amino acids in albescent and green leaves were analyzed by AccQ.Tag pre-column derivative method according to Waters Company. The results showed that both albescent and green leaves contained 18 kinds of amino acids, but the total amount of amino acids and the content of each amino acid were different between two stages. The content of amino acids in albescent leaves was 5.93%, and it's 2.12 times higher than that of green leaves. The more tender the tea leaves was, the higher the amino acid level would be. There was a positive correlation between the content of L-Theanine and the total amount of amino acids in Anji Baicha. The content of L-Theanine in albescent leaves was 3.33%, and it's 2.31 times higher than that of green leaves. The results also indicated that AccQ.Tag pre-column derivative HPLC method established by this research could well separate the amino acid components of tea plant, so it can be uesd to analyze and compare the amount of amino acids in different tea samples.
In order to separate the differentially expressed genes between albescent and green leaves of Anji Baicha, a subtracted cDNA library was constructed by the SSH method. According to the preliminary research from genetic level, Anji Baicha with high amino acid characteristic might trace to serine-glyoxylate aminotransferase gene and serine hydroxymethyl transferase gene. To reveal the miracle fact of amino acids enrichment in albino leaves, the further studies about these two genes are necessary and going on.
Theanine synthase can convert L-glutamic acid and ethylamine into L-theanine while ATP providing energy. In this study, the full-length cDNA of theanine synthase gene was cloned. The cDNA sequence has the full-length of 1503 bp, and with an Open Reading Frame of 1071 bp. It can encode 356 amino acids. Bioinformatics analysis showed that theanine synthase has 356 amino acid residues; has a theoretical molecular weight of 39 250.3 Da and theoretical PI value of 5.79. Bioinformatics prediction showed that this protein is hydrophilic and located not within the transmembrane; there are 26 phosphorylation sites within the polypeptied chain. The signal peptide analysis showed that there is no signal peptide and no winded helix domain present within the sequence; the protein is non-secreted and it functions in the cytoplasm. This reported result about cloning full-length cDNA of theanine synthase gene would be beneficial for using biotechnology to improve tea plant variety resources and regulate enhanceing L-theanine level.
The function of theanine synthase gene was preliminarily confirmed by prokaryotic expression and in vitro enzyme test. Via construction plasmid pMD 19-T-TS, the final recombinant plasmid pET-32a (+)-TS was constructed, then it was transfered to the expression vector Escherichia coli BL21. Afterward it was induced by IPTG to express recombinant protein. Result showed that a recombinant protein was produced with a molecular weight of about 59 kD, which was accordant with the predicted result. By in vitro enzyme test and HPLC analysis, the recombinant protein successfully catalyzed the synthesis of L-theanine.
The full-length cDNA of CDC48 gene in Anji Baicha was cloned by SMART-RACE. The cDNA sequence has the full-length of 2859 bp, and with an Open Reading Frame of 2 424 bp. It can encode 807 amino acids. Bioinformatics analysis showed that CDC48 has 807 amino acid residues; has a theoretical molecular weight of 89 901.9 Da and theoretical PI value of 5.16. Bioinformatics prediction showed that this protein is hydrophilic and located not within the transmembrane; there are 38 phosphorylation sites distributing unevenly in the polypeptied chain. The signal peptide analysis showed that there is no signal peptide but there are three possible winded helix domains present within the amino acid sequence; the protein is non-secreted and it functions in the nucleus. Researches showed that CDC48 is a spindle decomposition related protein, and relates to protein biodegradation pathway relying on ubiquitin; and CDC48 gene is a vital element participating in cell cycle regulation. From this point of view, cloning full-length cDNA of CDC48 gene and analyzing the structure & function of its protein are meaningful for unfolding the molecular mechanism of amino acids enrichment in albino leaves of Anji Baicha.
To analyze the differential expression of TS gene and CDC48 gene in albescent and green leaves, relative quantitation real-time PCR was applied. The results showed that the expression level of both TS and CDC48 genes were higher in albescent leaves than that of green leaves. TS catalyzes the synthesis of L-thenine and CDC48 is related to protein degradation into amino acids, so the over expression of these two genes in albino leaves could contribute to the property of high amino acid level during the albescent stage of Anji Baicha.
1.采用Waters公司AccQ.Tag柱前衍生高效液相色谱法对安吉白茶全白期与全绿期叶片中的氨基酸组分及含量进行检测,结果表明,安吉白茶的全白期与全绿期叶都含有包括茶氨酸在内的18种氨基酸,但两个时期氨基酸各组分的含量存在较大差异,安吉白茶全白期一芽二叶中的氨基酸含量达到5.93%,是全绿期一芽二叶中的2.12倍。且相同时期一芽二叶中的氨基酸含量高于第三、四叶中的氨基酸含量。茶氨酸含量与氨基酸总量呈正相关,安吉白茶全白期叶中的茶氨酸含量为3.33%,是全绿期叶中茶氨酸含量的2.31倍。本研究还表明AccQ.Tag柱前衍生高效液相色谱法能较好的分离茶树中的各氨基酸组分,有利于对不同样品进行分析与比较。
2.以安吉白茶全白期与全绿期叶为研究材料,采用SSH技术,成功构建了安吉白茶全白期与全绿期叶片的抑制消减杂交cDNA文库,筛选出了一批差异表达基因,并对相关差异表达基因进行了初步分析,发现丝氨酸-乙醛酸转氨酶基因,丝氨酸羟甲基转移酶基因可能与安吉白茶高氨基酸性状相关,因而有进一步研究的价值,其后续研究正在进行中,可望为阐明安吉白茶全白期氨基酸超常富集的分子机理提供重要信息。
3.茶氨酸合成酶在反应底物L-谷氨酸和乙胺的存在下,由ATP提供能量,能催化合成茶氨酸。本试验利用SMART RACE技术首次成功克隆了安吉白茶茶氨酸合成酶基因(TS)的全长cDNA序列。TS基因的cDNA全长为1503bp, ORF为1071 bp,编码356个氨基酸。经相关生物信息学分析,TS的氨基酸残基数为356,分子量为39250.3(Da),理论等电点(PI)为5.79,属于亲水性蛋白,TS蛋白不存在信号肽序列,属于非分泌蛋白,非跨膜蛋白,其磷酸化位点有26个。其氨基酸序列中可能不存在卷曲螺旋结构。通过亚细胞定位预测,安吉白茶TS蛋白是一种细胞质蛋白。克隆茶树茶氨酸合成酶基因的全长cDNA,对于利用生物技术手段改良茶树品种、以调控提高茶氨酸含量具有重要意义。
4.采用原核表达和体外酶试验法,对茶氨酸合成酶基因的功能进行了初步验证。通过中间质粒pMD 19-T-TS,成功构建了茶树TS蛋白的重组质粒pET-32a(+)-TS,并转入表达载体BL21,经IPTG诱导实现了重组蛋白的表达,且表达蛋白的分子量与预测的一致,约为59 kD。采用L-谷氨酸和乙胺为底物,利用重组蛋白进行体外酶催化试验,经HPLC检测表明,重组蛋白成功催化了茶氨酸的合成。
5.利用SMART RACE技术首次成功克隆了安吉白茶纺锤体分解相关蛋白基因(CDC48)的全长cDNA序列。其cDNA全长为2859bp, ORF为2424 bp,共编码807个氨基酸。经相关生物信息学分析表明,CDC48的氨基酸残基数为807,分子量为89901.9(Da),理论等电点(PI)为5.16,属于亲水性蛋白,CDC48不存在信号肽序列,属于非分泌蛋白,非跨膜蛋白,不发生跨膜运动,磷酸化位点有38个,它们不均匀分布于整个多肽链中,其氨基酸序列有三个区域可能形成卷曲螺旋。通过亚细胞定位预测,认为安吉白茶CDC48蛋白是一种细胞核蛋白,主要定位在核区。CDC48是参与细胞周期调控的重要元素,研究表明,CDC48与泛素依赖蛋白降解途径有关,所以克隆CDC48基因的全长cDNA,研究其蛋白质的结构和功能,对于探明安吉白茶阶段性返白过程中氨基酸超常富集的分子机理具有重要意义。
6.采用相对定量qPCR技术,以茶树18s rRNA为内参基因,以安吉白茶全绿期叶为对照组,对安吉白茶全白期TS基因和CDC48基因的相对表达水平进行了定量分析,结果表明TS基因和CDC48基因在安吉白茶白期叶中的表达水平比绿期叶的。TS催化L-谷氨酸和乙胺生成茶氨酸；而CDC48与泛素依赖蛋白降解途径有关,其在安吉白茶全白期叶中表达水平较高,可能与蛋白质降解,氨基酸富集相关。因此,这两个基因在安吉白茶全白期叶中表达水平高于绿期叶,可能与该时期氨基酸含量较高的性状相关。
Tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important economy crops. It is an evergreen bush and has over 3000 year history as a cultivation crop in China. Anji Baicha is a kind of specific variety with reversible albinism in spring shoots. During the albescent stage, the content of chlorophyll in albino leaves sharply reduces, but the amount of amino acids raise significantly. The amount of amino acids is about 2 times higher in albino leaves than that of normal leaves from ordinary varieties. L-Theanine is a characteristic ingredient of tea plant, and a non-protein amino acid, which accounts for 50～60% of total amount of amino acids in tea plant. L-Theanine is a kind of secondary metabolite with important physiology activities. Our previous research had isolated a bunch of differentially expressed genes related to the property of high amino acid level during stage albescent process of Anji Baicha by SSH and cDNA-AFLP. In this study, the full-length cDNA of two genes were cloned and their functions were further analyzed. The research results would support to regulate enhancing of the L-theanine level in tea plant from both theoretical and practical aspects. The main results were as follows:
The components and contents of amino acids in albescent and green leaves were analyzed by AccQ.Tag pre-column derivative method according to Waters Company. The results showed that both albescent and green leaves contained 18 kinds of amino acids, but the total amount of amino acids and the content of each amino acid were different between two stages. The content of amino acids in albescent leaves was 5.93%, and it's 2.12 times higher than that of green leaves. The more tender the tea leaves was, the higher the amino acid level would be. There was a positive correlation between the content of L-Theanine and the total amount of amino acids in Anji Baicha. The content of L-Theanine in albescent leaves was 3.33%, and it's 2.31 times higher than that of green leaves. The results also indicated that AccQ.Tag pre-column derivative HPLC method established by this research could well separate the amino acid components of tea plant, so it can be uesd to analyze and compare the amount of amino acids in different tea samples.
In order to separate the differentially expressed genes between albescent and green leaves of Anji Baicha, a subtracted cDNA library was constructed by the SSH method. According to the preliminary research from genetic level, Anji Baicha with high amino acid characteristic might trace to serine-glyoxylate aminotransferase gene and serine hydroxymethyl transferase gene. To reveal the miracle fact of amino acids enrichment in albino leaves, the further studies about these two genes are necessary and going on.
Theanine synthase can convert L-glutamic acid and ethylamine into L-theanine while ATP providing energy. In this study, the full-length cDNA of theanine synthase gene was cloned. The cDNA sequence has the full-length of 1503 bp, and with an Open Reading Frame of 1071 bp. It can encode 356 amino acids. Bioinformatics analysis showed that theanine synthase has 356 amino acid residues; has a theoretical molecular weight of 39 250.3 Da and theoretical PI value of 5.79. Bioinformatics prediction showed that this protein is hydrophilic and located not within the transmembrane; there are 26 phosphorylation sites within the polypeptied chain. The signal peptide analysis showed that there is no signal peptide and no winded helix domain present within the sequence; the protein is non-secreted and it functions in the cytoplasm. This reported result about cloning full-length cDNA of theanine synthase gene would be beneficial for using biotechnology to improve tea plant variety resources and regulate enhanceing L-theanine level.
The function of theanine synthase gene was preliminarily confirmed by prokaryotic expression and in vitro enzyme test. Via construction plasmid pMD 19-T-TS, the final recombinant plasmid pET-32a (+)-TS was constructed, then it was transfered to the expression vector Escherichia coli BL21. Afterward it was induced by IPTG to express recombinant protein. Result showed that a recombinant protein was produced with a molecular weight of about 59 kD, which was accordant with the predicted result. By in vitro enzyme test and HPLC analysis, the recombinant protein successfully catalyzed the synthesis of L-theanine.
The full-length cDNA of CDC48 gene in Anji Baicha was cloned by SMART-RACE. The cDNA sequence has the full-length of 2859 bp, and with an Open Reading Frame of 2 424 bp. It can encode 807 amino acids. Bioinformatics analysis showed that CDC48 has 807 amino acid residues; has a theoretical molecular weight of 89 901.9 Da and theoretical PI value of 5.16. Bioinformatics prediction showed that this protein is hydrophilic and located not within the transmembrane; there are 38 phosphorylation sites distributing unevenly in the polypeptied chain. The signal peptide analysis showed that there is no signal peptide but there are three possible winded helix domains present within the amino acid sequence; the protein is non-secreted and it functions in the nucleus. Researches showed that CDC48 is a spindle decomposition related protein, and relates to protein biodegradation pathway relying on ubiquitin; and CDC48 gene is a vital element participating in cell cycle regulation. From this point of view, cloning full-length cDNA of CDC48 gene and analyzing the structure & function of its protein are meaningful for unfolding the molecular mechanism of amino acids enrichment in albino leaves of Anji Baicha.
To analyze the differential expression of TS gene and CDC48 gene in albescent and green leaves, relative quantitation real-time PCR was applied. The results showed that the expression level of both TS and CDC48 genes were higher in albescent leaves than that of green leaves. TS catalyzes the synthesis of L-thenine and CDC48 is related to protein degradation into amino acids, so the over expression of these two genes in albino leaves could contribute to the property of high amino acid level during the albescent stage of Anji Baicha.
引文
[1]茶树种质资源与遗传改良[M].中国农业科学技术出版社,2006.
[2]Sakato Y. Studies on the chemical constituents of tea. Part Ⅲ. On a new amide theanine [J]. Nippon Nogeikagaku Kaishi.1949,23:262-267.
[3]Casimir J, Jadot J. Separation and characterization of N-ethyl-gamma-glutamine from Xerocomus badius [J]. Biochimica et biophysica acta.1960,39:462.
[4]Tsushida T, Takeo T. Occurrence of theanine in Camellia japonica and Camellia sasanqua seedlings [J]. Agricultural and Biological Chemistry.1984,48(11):2861-2 862.
[5]Deng W W, Ogita S, Ashihara H. Distribution and biosynthesis of theanine in Theaceae plants [J]. Plant Physiology and Biochemistry.2009.
[6]Ekborg-Ott K H, Taylor A, Armstrong D W. Varietal differences in the total and enantiomeric composition of theanine in tea[J]. J. Agric. Food Chem.1997,45(2): 353-363.
[7]宛晓春.茶叶生物化学[M].北京：中国农业出版社,2003,34-35.
[8]Arai M, Matsuura T, Kakuda T. Variation of the yields and the chemical composition in leaves on nutriculture of tea plant [J]. Nippon Dojo Hiryogaku Zasshi.1989,60: 157-159.
[9]王泽农.茶叶生物化学[M].北京：农业出版社,1980,48-56.
[10]Deng W W, Ogita S, Ashihara H. Distribution and biosynthesis of theanine in Theaceae plants [J]. Plant Physiol Biochem.2010,48(1):70-72.
[11]Kito M, Kokura H, Izaki J, et al. Theanine, a precursor of the phloroglucinol nucleus of catechins in the tea plants [J]. Phytochemistry.1968,7(4):599-603.
[12]李素芳,成浩,虞富莲,等.安吉白茶阶段性返白过程中氨基酸的变化[J].茶叶科学,1996,16(2)：153-154.
[13]成浩,李素芳,陈明,等.安吉白茶特异性状的生理生化本质.茶叶科学,1999,19(2)：87-92.
[14]成浩,陈明,虞富莲,等.茶叶片阶段性返白过程中色素蛋白复合体的变化.植物生理学通讯,2000,36(4)：300-304.
[15]李素芳,陈树尧,成浩.茶树阶段性返白现象的研究.叶绿体超微结构的变化.茶叶科学,1995,15(1)：23-26.
[16]李素芳,成 浩,虞富莲等.白茶返白复绿过程中的细胞学研究.中国植物生理学会第七次全国会议论文,太原,144.
[17]郭雅敏.安吉白茶的性状与发展前景[J].茶叶,1997,23(4)：23-24.
[18]郭亮,沈微,王正祥,等.生物转化法生产茶氨酸的重组大肠杆菌的构建[J].食品与生物技术学报.2005,24(02)：1673-1689.
[19]王贤波,王丽鸳,成 浩,等.茶氨酸生物合成工程菌构建[J].茶叶科学.2007(01)：61-66.
[20]成浩,王丽鸳,周 健,等.固定化基因工程菌生物合成茶氨酸的方法：中国,200810162598.9[P].2009,0603.
[21]Sasaoka K, Kito M, Y O, et al. Some properties of the theanine synthesizing enzyme in tea seedlings [J]. Bio Chem.1965,29(11):984-988.
[22]Kimura T, Sugahara I, Hanai K, et al. Purification and characterization of γ-glutamylmethylamide synthetase from Methylophaga sp. AA-30[J]. Biosci Biotechnol Biochem,1992,56(5):708-711.
[23]立木隆,冈田幸隆,小关诚,等.茶氨酸的制造方法：日本,PCT/JP2003/005077[P].2004-2-26.
[24]Abelian H V, Okubo T, Shamtsian M M, et al. A novel method of production of theanine by immobilized Pseudomonas nitroreducens cells [J]. Biosci Biotechnol Biochem.1993,57(3):481-483.
[25]江波,张涛,帅玉英.天然茶氨酸的制备方法：中国,200810021187.8[P].2009-01-14.
[26]Suzuki H, Kumagai H, Tochikura T. y-Glutamyltranspeptidase from Escherichia coli K-12:purification and properties [J]. J Bacteriol.1986,168:1325-1331.
[27]Suzuki H, Kumagai H, T E, et al. Molecular cloning of Escherichia coli K-12 ggt and rapidisolation of y-glutamyltranspeptidase [J]. Biochem Biophys Res.1988,150: 33-38.
[28]Sachiko Y, Kousuke U, Mamoru W, et al. Purification and characterzation of glutamine synthetase an enzyme usable for production of theanine [J]. Biosci Biotechol Biochem.2004,69(8):1888-1897.
[29]Sachiko Y, Kousuke U, Mamoru W, et al. Theanine production by couple fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells [J]. Biosci Biotechol Biochem.2005,69(4): 784-789.
[30]Matsuura T, Kakuda T, Kinoshita T. Theanine formation by tea suspension cell [J]. Biosci Biotech Biochem.1992,56(8):1179-1181.
[31]Matsuura T, Kakuda T, Kinoshita T, et al. Effects of Plant Growth Regulators and Carbon Sources on Theanine Formation in Callus Cultures of Tea (Camellia Sinensis) [J]. Biosci Biotech Biochem.1994,58(8):1519-1521.
[33]余继红,华东,华萍,等.大规模悬浮培养茶叶细胞合成茶氨酸培养基组成优化研究[J].茶叶科学.2006(02)：131-135.
[34]陈瑛,钟俊辉.茶细胞悬浮培养生产茶氨酸的工艺条件研究[J].绍兴文理学院学报.1998,18(05)：71-75.
[35]李健.牛肝菌液态发酵生成茶氨酸的研究[D].安徽：安徽农业大学硕士学位论文,2007.
[36]Lichtenstein N. Preparation of y-alkylamides of glutamic acid [J]. J Am Chem Soc. 1942,64:1021-1022.
[37]桥爪斌.茶氨酸的化学合成[J].日本农业化学会志.1951(25)：25-26.
[38]Yamada Y. The chemical synthesis of L2theanine [J]. Bull. Chem. Soc. Jap.1966, 39:1999-2000.
[39]李炎,王秀芬,刘锋.茶氨酸合成：中国,200310117502.4[P].2004-12-15.
[40]郑国斌.茶氨酸的制备方法：中国,200410041298.7[P].2005-12-14.
[41]陈新,王凯文.一种L-茶氨酸的改进合成方法：中国,200610092083.7[P].2009-01-14.
[42]Setsuko A, Takami T. Method for producing theanine JP278848[P].2001-10-10.
[43]Yan S H, Jean-Pierre D, Marc M.高纯度茶氨酸的合成与特性(英文)[J].茶叶科学.2003,23(02)：99-104.
[44]郭丽芸,刘毅,贾晓娟,等.酶法拆分DL-茶氨酸及其分离纯化[J].茶叶科学.2006(01)：31-36.
[45]Hirokazu K. Production oftheanine:日本, JP5070419[P].1993-03-23.
[46]Setsuko A, Takami T, Akio S. Production of theanine JP026383[P].2000-01-25.
[47]陈瑛.茶氨酸提取方法的研究[J].绍兴文理学院学报.1997(06)：71-76.
[48]林智,杨勇,谭俊峰,等.茶氨酸提取纯化工艺研究[J].天然产物研究与开发.2004(05)：67-72.
[49]袁 华,高小红,闫志国,等.离子交换法从茶叶中提取茶氨酸[J].精细石油化工进展.2006(03)：48-50.
[50]久保田裕司,泷原效宣.茶氨酸的制造方法以及茶氨酸高营养食品：中国,200310100097.5[P].
[51]肖伟涛,朱小兰,陈波,等.制备高效液相色谱分离纯化茶氨酸对照品[J].中草药.2004(02)：34-36.
[52]陈波,姚守拙,朱小兰,等.茶氨酸标准对照品的分离制备工艺：中国,03118181.3[P].2004-09-29.
[53]Ying Z, Bo C, Zhiqiang H. Preparative Isolation and Purification of L-Theanine by HPLC [J]. J Liq Chromatogr & Related Technol.2004,27(5):875-884.
[54]王吉村,药立波,赵忠良.筛选差异基因和蛋白质的方法进展[J].生物化学与生物物理进展,2001,28(1)：33-36
[55]黄薇,方孝东,赵文明,等.分离差异表达基因的方法[J].生物工程学报,2002,18(7)：522-524
[56]Liang P, Arthur BP. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992,257:967-970
[57]方春媛,陈年来,任晓燕,等.水杨酸诱导的甜瓜DDRT-PCR分析及其基因差异表达片段的分离[J].甘肃农业大学学报,2008,43(2)：90-95
[58]马月萍,戴思兰mRNA差别显示技术及其在植物基因研究中的应用[J].北京林业大学学报,2003,25(2)：81-84
[59]刘国花.mRNA差异显示技术及其在植物基因工程中的应用[J].重庆文理学院学报,2006,5(1)：63-65
[60]李风兰,王淑菊mRNA差异显示技术研究进展[J].黑龙江农业科学,2007,1(1)：86-89
[61]陈清轩mRNA差异显示技术研究进展[J].农业生物技术学报,2004,12(5),597-601
[62]杨新艳,刘培欣,单文鲁,等mRNA差异显示技术及其改进[J].动物医学进展,2004,25(6)：19-21
[63]刘永军,景建洲,贾敬芬,等.用mRNA差异显示技术分离盐胁迫下小麦耐盐相关cDNA[J].西北大学学报,2006,36(1)：89-91
[64]孔凡晶,陈孝,马有志,等.利用差异显示技术克隆小麦抗白粉病相关基因的研究[J].麦类作物学报,2004,24(1)：11-14
[65]黄贵修.利用差异显示技术探索橡胶树死皮病病因的研究[D].华南热带农业大学,1999,16(3)：378-382
[66]陈永华,赵森,严钦泉,等.利用差异显示法研究水稻耐淹涝相关基因[J].农业生物技术学报,2006,14(6)：894-898
[67]湛凤凰.代表性差异分析方法在基因克隆中的应用及其发展[J].国外医学遗传学分册,1998,21(3)：113-116
[68]方孝东,林栖凤,李冠一.对代表性分析方法的改进[J].海南大学学报自然科学版,2003,21(2)：135-138
[69]杨 林,袁爱力,李勇,等.一种基因克隆的新策略一代表性差异分析法[J].国外医学遗传学分册,1998,21(3)：113-116
[70]Diatchenko L, Lau Y F, Campbell A P, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes [J]. Proc Natl Acad Sci USA,1996,93:6025-6030
[71]赵永贞,蒋伟明,陈秀荔,等.抑制性消减杂交技术在对虾基因克隆中的应用[J].动物医学进展,2008,29(4)：76-80
[72]李 茂,字学娟,蒋昌顺.抑制消减杂交在热带作物研究中的应用[J].亚热带农业研究,2008,4(2)：151-153
[73]Money T, Reader S, Qu L J, et al. AFLP-based mRNA fingerprinting [J]. Nucleic Acids Research,1996 (24):2616-2617
[74]Donson J, Fang Y, Espiritu Santo G, et al. Comprehensive gene expression analysis by transcript profiling [J]. Plant Molecular Biology,2002 (48):75-97
[75]李驰,卢新雄,陈晓玲,等.利用cDNA-AFLP技术分析老化棉花种子基因差异表达[J].棉花学报,2007,19(4)：255-260
[76]李凌,宋文芹,毛英伟,等.用cDNA-AFLP银染技术研究与花椰菜花色相关的基因.南开大学学报(自然科学版),2000,33(4)：33-36
[77]刘涛,徐平珍,胡运乾,等.板栗花序与叶的cDNA-AFLP的差异表达分析[J].中国农学通报,2007,23(2)：81-84
[78]余梅,江昌俊,叶爱华,等.利用cDNA-AFLP技术研究茶树花蕾发育基因差异表达片段[J].茶叶科学,2007,27(3)：259-264
[79]Mamati G. E. Tea polyphenols biosynthesis and genes expression control in tea (Camellia sinensis),浙江大学博士学位论文,2005
[80]陈桂平,黄占景,马闻师,等.盐胁迫下小麦耐盐突变体后代差异cDNA的分离和鉴定.中国农业科学,2003,36(9)：996-999
[81]陈桂平,马闻师,黄占景,等.小麦耐盐突变体盐胁迫下SIR73基因片段的分离和鉴定.遗传,2003,25(2)：173-176
[82]张莉,江昌俊,胥振国,等.用cDNA-AFLP技术研究茶树种子在低温贮藏过程中差异基因的表达[J].安徽农业大学学报,2008,35(3)：319-323
[83]吴扬.应用cDNA-AFLP技术分离安吉白茶阶段性返白过程的差异表达基因[D].湖南农业大学硕士学位论文,2009.
[84]丁友成,张辉,钟明安,等.基因芯片技术筛选大肠癌肝转移差异表达基因[J].南京医科大学学报(自然科学版),2008,28(4)：476-479
[85]汤玉瑜,陈永文,费蕾,等.基因芯片筛选HepG2与HepG2.2.15细胞中的差异表达基因[J].免疫学杂志,2008,24(3)：329-333
[86]蒋立坚,赖仁发,李卫国,等.基因芯片筛选成釉细胞瘤差异表达基因的研究[J].中国病理生理杂志,2008,24(5)：992-996
[87]李沛,凌志强,赵继敏,等.建立cDNA微矩阵基因芯片技术并分析食管癌细胞系Eca109基因表达谱[J].生物医学工程学杂志,2008,25(2)：412-417
[88]王冰林,柳俊,李媛媛,等cDNA微阵列鉴定差异表达基因的可靠性分析[J].华北农学报,2008,23(3)：5-8
[89]韩杨云,曾义.生物芯片技术与胶质瘤基因的差异表达[J].国际神经学与神经外科学,2007,34(6)：568-571
[90]徐清华.基因芯片技术的研究进展[J].中国优生与遗传杂志,2007,15(1)：13-14
[91]Deng W W, Ogita S, Ashe K H. Biosynthesis of theanine (γ-ethylamino-1-glutamic acid) in seedlings of Camellia sinensis [J]. Phytochemistry Letters.2008,1(2): 115-119.
[92]杨贤强,王岳飞,陈留记,等.茶多酚化学[M].上海：上海科学技术出版社,2003.
[93]Martens S, Knott J, Seitz CA, et al. Impact of biochemical pre-studies on specific engineering strategies of flavonoid biosynthesis in plant tissues [J]. Biochemical Engineering Journal,2003,14:227-235.
[94]Matsumoto S, Takeuchi A, Hayatsu M, et al. Molecular cloning of phenylalanine ammonium lyase cDNA and classification of varieties and cultivars of tea plants (Camellia sinensis)using a tea PAL cDNA probe [J]. Theor. Appl. Genet.,1994,89: 671-675.
[95]Takeuchi A, Matsumoto S, Hayatsu M. Chalcone synthase from Camellia sinensis: isolation of the cDNA and the organspecific and sugar responsive expression of the genes [J]. Plant Cell Physiol,1994(5):1011-1018.
[96]Takeuchi A, Matsumoto S. Dihydroflavonol 4-reductase mRNA from Camellia sinensis. Http://www.ncbi.Nlm.Nih.Gov,Accessing No.AB018685.
[97]Singh K, Kumar S, Ahuja P S. Cloning and characterization of cDNA encoding transcinnamate 4-hydroxylase from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.AY641731.
[98]Rani A, Kumar S, Ahuja PS. Camellia sinensis 4-Coumaroyl CoA Ligase mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing.No.DQ194356.
[99]Rani A, Kumar S, Ahuja PS. Camellia sinensis Cultivar Upasi-10 Chalcone Isomerasc (CHI) mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No. DQ120521.
[100]Chen L, Ma CL, Zhao LP. Molecular cloning of full-length cDNA of the chalcone isomerase(CHI) gene from Camellia sinensis (L.) O. Kuntze. Http://www.Ncbi.Nlm. Nih.Gov,Accessing No.DQ904329.
[101]Singh K, Kumar S, Ahuja PS.2004 Molecular cloning of full length cDNA of flavanone 3-hydroxylase(f3h) gene from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.Ncbi.Nlm.Nih.Gov,2004,Accessing No.AY641730.
[102]Rani A, Kumar S, Ahuja PS. Camellia sinensis Flavonoid 3',5'-Hydroxylasc mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.DQ194358.
[103]Park J S,Kim J B, Kim Y H. Camellia sinensis leucoanthocyanidin reductase (LCR) mRNA. Http://www.ncbi.Nlm.Nih.Gov,Accessing No.AY169404.
[104]Singh K, Kumar S, Ahuja PS. Cloning of full length cDNA encoding anthocyanidin synthase (ANS) from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www. ncbi.Nlm.Nih.Gov,Accessing No.AY830416.
[105]Singh K, Kumar S, Ahuja PS. Molecular cloning of full length cDNA of leucoanthocyanidin reduetase(LAR) gene from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.ncbi.Nlm.Nih.Gov, Accessing No. AY641729.
[106]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.
[107]Kato M, Mizuno K, Fujimura T, el al. Purification and Characterization of Caffeine Synthase from Tea Leaves [J]. Plant Physiology,1999,120:579-586.
[108]张广辉,梁月荣,吴颖.咖啡碱生物合成研究进展及在茶树育种中的应用[J].茶叶,2005,31(1)：18-23.
[109]吴颖,梁月荣.S-腺苷甲硫氨酸在茶树生理代谢中的研究现状[J].茶叶,2005,31(2)：85-87.
[110]冯艳飞,梁月荣,茶树S-腺苷甲硫氨酸合成酶基因的克隆和序列分析[J].茶叶科学,2001,21(1)：21-25
[111]Kato M. Biochemistry and molecular biology in caffeine biosynthesis-Molecular cloning and gene expressing of caffeine synthase. In:Proceedings of 2001 International Conference on O-Cha (Tea) Science (SessinⅡ),21-24, Shizuoka, Japan.
[112]Mizutani M, Nakanishi H, Ema J, et al. Cloning of β-primeverosidase from tea Leaves, a key enzyme in tea aroma formation [J]. Plant Physiology,2002,130: 2164-2176.
[113]Jiang CJ, Yang SL, Li YI-I, el al. Camellia sinensis beta-1,3-glucosidase-like mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.AF537127.
[114]李远华,江昌俊,余有本.茶树p-葡萄糖苷酶基因mRNA的表达[J].南京农业大学学报,2005,28(2)：103-106.
[115]Rawat R, Gulati A, Hallan V, el al. Molecular characterization of beta-glucosidase from regional Kangra tea clone (Camellia sinensis (L.)O Kuntze). AM285295.
[116]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.
[117]王朝霞,江昌俊,蔡海云.茶树p-1,3-葡聚糖酶基因cDNA片段的克隆与序列分析[J].安徽教育学院学报,2006,24(3)：67-70.
[118]Singh K, Kumar S, Ahuja PS. Camellia sinensis HSP70 (hsp70) mRNA (partial cd). Http://www. Ncbi. Nlm. Nih. Gov, Accessing No. AY694190.
[119]Singh K, Kumar S, Ahuja PS. Cloning and sequencing of catalase gene from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.Ncbi.Nlm.Nih.Gov, Accessing No.AY641732.
[120]Singh K, Vyas D, Kumar S, el al. Cloning of cDNA encoding manganese superoxide dismutase (MnSOD) from Camellia sinensis (L.) O. Kuntze. Http:www. Ncbi.Nlm.Nih.Gov,Accessing No.AY641734.
[121]Singh K, Kumar S, Ahuja PS. Camellia sinensis Cu/Zn superoxide dismutase mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.AY694187.
[122]Tomimoto Y, Ikehashi H, Kakeda K, el al. A pistil-specific PR-1 like protein of Camellia, its expression, sequence and genealogical position [J]. Breeding Sci,1999, 49:97-104.
[123]Paul A, Kumar S, Ahuja PS. Camellia sinensis metallothionin 1 mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.DQ442270.
[124]Paul A, Kumnr S, Ahuja PS. Camellia sinensis metallothionin 2 mRNA. Http://www.ncbi.him.nih.Gov,Accessing No.DQ442271.
[125]Paul A, Kumar S, Ahuja PS. Camellia sinensis ascorbate peroxidase mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.DQ442272.
[126]Chen L, Zhang YL, Zhao LP. Molecular cloning of full-length cDNA of the ACC oxidase gene from Camellia sinensis (L.) O. Kuntze. Http://www.Ncbi.Nlm.Nih. Gov, Accessing No.DQ904328.
[127]Frohman M A, Dush M K, Martin G R. Rapid production of full-length cDNAs from rare transcripts:Amplification using a single gene-specific oligonucleotide primer [J]. Proc Natl Acad Sci USA,1988,85:8998-9002.
[128]王关林,方宏筠.植物基因工程原理与技术[M].北京：科学出版社,1998158-161.
[129]赵炜,郑佐华,毛裕民.全长cDNA的获得RACE及其他方法进展[J].生命科学,1999,11(2)：92-96.
[130]明洪,黄秉仁RACE-cDNA末端快速扩增技术进展[J].生物工程进展,1997,17(5)：7-12.
[131]张庆华,茅矛,陈竺.基因组研究中全长cDNA克隆的策略[J].生物工程进展,2000,20(4)：3-5.
[132]孔凡晶,马有志,陈孝,等.差异显示和RACE技术的发展与应用[J].生物技术通报,2003,1(4)：13-16.
[133]陈启龙RACE技术的研究进展及其应用[J].黄山学院学报,2006,8(3)：95-98.
[134]LIU X W, GOROVSKY M A. Mapping the 5'and 3'ends of Tetrahymena thermophila mRNA using RNA ligase mediated amplification of cDNA ends(RLM-RACE)[J]. Nucleic Acids Res,1993,21(21):4954-4960.
[135]Maruyama K, Sugano S. Oligo-Capping—A simple method to replace the CAP structure of Eukaryotic messeger-RNAs with oligoribonucleotides [J]. Gene,1994, 138:171-174.
[136]周维一,黄翠琴,王寿昆,等.RACE一种研究新基因的有效方法[J].热带医学杂志,2007,7(4)：386-389.
[137]SCHAEFER BC. Revolutions in rapid amplification of cDNA ends:new strategies for polymerase chain reaction cloning of full-length cDNA ends [J]. Anal Biochem, 1995,227(2):255-273.
[138]Ye Z, Connor JR. cDNA cloning by amplification of circularized first strand cDNAs reveals non-IRE-regulated iron-responsive mRNAs [J]. Biochem Biophys Res Commun,2000,275(1):223-227.
[139]邓雪柯,殷建华,曹毅.3种5'RACE技术的比较与优化[J].成都医学院学报,2007,2(1)：20-25.
[140]董志敏,李英慧,张宝石,等.一种获得大片段克隆的SMART全长cDNA文库构建方法[J].大豆科学,2007,12(3)：223-226.
[141]汤皴.茶氨酸的合成药理及其在食品中的应用.茶叶通报,2002,24(4)：19-21.
[142]陈宗懋.茶氨酸具有降压功能.中国茶叶,1997,2：27.
[143]丁王,丁雪.茶氨酸的抗疲劳作用研究.中国公共卫生,2002,18(3)：315-317.
[144]谭和平,陈丽,叶善蓉,等.茶叶中氨基酸的检测方法概述[J].中国检测技术,2007,33(6)：1-4.
[145]Liepman AH, Olsen LJ. Peroxisomal alanine:glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana [J]. Plant J,2001;25:487-498.
[146]Daisuke Igarashi, Hiroko Tsuchida, Mitsue Miyao, et al. Glutamate:Glyoxylate Aminotransferase Modulates Amino Acid Content during Photorespiration [J]. Plant Physiol,2006,142:901-910
[147]刘丽娜,刘祥林,陈志玲.细胞周期调控的一个重要元素-CDC48[J].生物技术通报,2006,5：26-29
[148]Frank Madeo, Jan Schlauer, Hans Zischka, et al. Tyrosine Phosphorylation Regulates Cell Cycle-dependent Nuclear Localization of Cdc48p[J]. Molecular Biology of the Cell,1998,9(1):131-141
[2]Sakato Y. Studies on the chemical constituents of tea. Part Ⅲ. On a new amide theanine [J]. Nippon Nogeikagaku Kaishi.1949,23:262-267.
[3]Casimir J, Jadot J. Separation and characterization of N-ethyl-gamma-glutamine from Xerocomus badius [J]. Biochimica et biophysica acta.1960,39:462.
[4]Tsushida T, Takeo T. Occurrence of theanine in Camellia japonica and Camellia sasanqua seedlings [J]. Agricultural and Biological Chemistry.1984,48(11):2861-2 862.
[5]Deng W W, Ogita S, Ashihara H. Distribution and biosynthesis of theanine in Theaceae plants [J]. Plant Physiology and Biochemistry.2009.
[6]Ekborg-Ott K H, Taylor A, Armstrong D W. Varietal differences in the total and enantiomeric composition of theanine in tea[J]. J. Agric. Food Chem.1997,45(2): 353-363.
[7]宛晓春.茶叶生物化学[M].北京：中国农业出版社,2003,34-35.
[8]Arai M, Matsuura T, Kakuda T. Variation of the yields and the chemical composition in leaves on nutriculture of tea plant [J]. Nippon Dojo Hiryogaku Zasshi.1989,60: 157-159.
[9]王泽农.茶叶生物化学[M].北京：农业出版社,1980,48-56.
[10]Deng W W, Ogita S, Ashihara H. Distribution and biosynthesis of theanine in Theaceae plants [J]. Plant Physiol Biochem.2010,48(1):70-72.
[11]Kito M, Kokura H, Izaki J, et al. Theanine, a precursor of the phloroglucinol nucleus of catechins in the tea plants [J]. Phytochemistry.1968,7(4):599-603.
[12]李素芳,成浩,虞富莲,等.安吉白茶阶段性返白过程中氨基酸的变化[J].茶叶科学,1996,16(2)：153-154.
[13]成浩,李素芳,陈明,等.安吉白茶特异性状的生理生化本质.茶叶科学,1999,19(2)：87-92.
[14]成浩,陈明,虞富莲,等.茶叶片阶段性返白过程中色素蛋白复合体的变化.植物生理学通讯,2000,36(4)：300-304.
[15]李素芳,陈树尧,成浩.茶树阶段性返白现象的研究.叶绿体超微结构的变化.茶叶科学,1995,15(1)：23-26.
[16]李素芳,成 浩,虞富莲等.白茶返白复绿过程中的细胞学研究.中国植物生理学会第七次全国会议论文,太原,144.
[17]郭雅敏.安吉白茶的性状与发展前景[J].茶叶,1997,23(4)：23-24.
[18]郭亮,沈微,王正祥,等.生物转化法生产茶氨酸的重组大肠杆菌的构建[J].食品与生物技术学报.2005,24(02)：1673-1689.
[19]王贤波,王丽鸳,成 浩,等.茶氨酸生物合成工程菌构建[J].茶叶科学.2007(01)：61-66.
[20]成浩,王丽鸳,周 健,等.固定化基因工程菌生物合成茶氨酸的方法：中国,200810162598.9[P].2009,0603.
[21]Sasaoka K, Kito M, Y O, et al. Some properties of the theanine synthesizing enzyme in tea seedlings [J]. Bio Chem.1965,29(11):984-988.
[22]Kimura T, Sugahara I, Hanai K, et al. Purification and characterization of γ-glutamylmethylamide synthetase from Methylophaga sp. AA-30[J]. Biosci Biotechnol Biochem,1992,56(5):708-711.
[23]立木隆,冈田幸隆,小关诚,等.茶氨酸的制造方法：日本,PCT/JP2003/005077[P].2004-2-26.
[24]Abelian H V, Okubo T, Shamtsian M M, et al. A novel method of production of theanine by immobilized Pseudomonas nitroreducens cells [J]. Biosci Biotechnol Biochem.1993,57(3):481-483.
[25]江波,张涛,帅玉英.天然茶氨酸的制备方法：中国,200810021187.8[P].2009-01-14.
[26]Suzuki H, Kumagai H, Tochikura T. y-Glutamyltranspeptidase from Escherichia coli K-12:purification and properties [J]. J Bacteriol.1986,168:1325-1331.
[27]Suzuki H, Kumagai H, T E, et al. Molecular cloning of Escherichia coli K-12 ggt and rapidisolation of y-glutamyltranspeptidase [J]. Biochem Biophys Res.1988,150: 33-38.
[28]Sachiko Y, Kousuke U, Mamoru W, et al. Purification and characterzation of glutamine synthetase an enzyme usable for production of theanine [J]. Biosci Biotechol Biochem.2004,69(8):1888-1897.
[29]Sachiko Y, Kousuke U, Mamoru W, et al. Theanine production by couple fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells [J]. Biosci Biotechol Biochem.2005,69(4): 784-789.
[30]Matsuura T, Kakuda T, Kinoshita T. Theanine formation by tea suspension cell [J]. Biosci Biotech Biochem.1992,56(8):1179-1181.
[31]Matsuura T, Kakuda T, Kinoshita T, et al. Effects of Plant Growth Regulators and Carbon Sources on Theanine Formation in Callus Cultures of Tea (Camellia Sinensis) [J]. Biosci Biotech Biochem.1994,58(8):1519-1521.
[33]余继红,华东,华萍,等.大规模悬浮培养茶叶细胞合成茶氨酸培养基组成优化研究[J].茶叶科学.2006(02)：131-135.
[34]陈瑛,钟俊辉.茶细胞悬浮培养生产茶氨酸的工艺条件研究[J].绍兴文理学院学报.1998,18(05)：71-75.
[35]李健.牛肝菌液态发酵生成茶氨酸的研究[D].安徽：安徽农业大学硕士学位论文,2007.
[36]Lichtenstein N. Preparation of y-alkylamides of glutamic acid [J]. J Am Chem Soc. 1942,64:1021-1022.
[37]桥爪斌.茶氨酸的化学合成[J].日本农业化学会志.1951(25)：25-26.
[38]Yamada Y. The chemical synthesis of L2theanine [J]. Bull. Chem. Soc. Jap.1966, 39:1999-2000.
[39]李炎,王秀芬,刘锋.茶氨酸合成：中国,200310117502.4[P].2004-12-15.
[40]郑国斌.茶氨酸的制备方法：中国,200410041298.7[P].2005-12-14.
[41]陈新,王凯文.一种L-茶氨酸的改进合成方法：中国,200610092083.7[P].2009-01-14.
[42]Setsuko A, Takami T. Method for producing theanine JP278848[P].2001-10-10.
[43]Yan S H, Jean-Pierre D, Marc M.高纯度茶氨酸的合成与特性(英文)[J].茶叶科学.2003,23(02)：99-104.
[44]郭丽芸,刘毅,贾晓娟,等.酶法拆分DL-茶氨酸及其分离纯化[J].茶叶科学.2006(01)：31-36.
[45]Hirokazu K. Production oftheanine:日本, JP5070419[P].1993-03-23.
[46]Setsuko A, Takami T, Akio S. Production of theanine JP026383[P].2000-01-25.
[47]陈瑛.茶氨酸提取方法的研究[J].绍兴文理学院学报.1997(06)：71-76.
[48]林智,杨勇,谭俊峰,等.茶氨酸提取纯化工艺研究[J].天然产物研究与开发.2004(05)：67-72.
[49]袁 华,高小红,闫志国,等.离子交换法从茶叶中提取茶氨酸[J].精细石油化工进展.2006(03)：48-50.
[50]久保田裕司,泷原效宣.茶氨酸的制造方法以及茶氨酸高营养食品：中国,200310100097.5[P].
[51]肖伟涛,朱小兰,陈波,等.制备高效液相色谱分离纯化茶氨酸对照品[J].中草药.2004(02)：34-36.
[52]陈波,姚守拙,朱小兰,等.茶氨酸标准对照品的分离制备工艺：中国,03118181.3[P].2004-09-29.
[53]Ying Z, Bo C, Zhiqiang H. Preparative Isolation and Purification of L-Theanine by HPLC [J]. J Liq Chromatogr & Related Technol.2004,27(5):875-884.
[54]王吉村,药立波,赵忠良.筛选差异基因和蛋白质的方法进展[J].生物化学与生物物理进展,2001,28(1)：33-36
[55]黄薇,方孝东,赵文明,等.分离差异表达基因的方法[J].生物工程学报,2002,18(7)：522-524
[56]Liang P, Arthur BP. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science,1992,257:967-970
[57]方春媛,陈年来,任晓燕,等.水杨酸诱导的甜瓜DDRT-PCR分析及其基因差异表达片段的分离[J].甘肃农业大学学报,2008,43(2)：90-95
[58]马月萍,戴思兰mRNA差别显示技术及其在植物基因研究中的应用[J].北京林业大学学报,2003,25(2)：81-84
[59]刘国花.mRNA差异显示技术及其在植物基因工程中的应用[J].重庆文理学院学报,2006,5(1)：63-65
[60]李风兰,王淑菊mRNA差异显示技术研究进展[J].黑龙江农业科学,2007,1(1)：86-89
[61]陈清轩mRNA差异显示技术研究进展[J].农业生物技术学报,2004,12(5),597-601
[62]杨新艳,刘培欣,单文鲁,等mRNA差异显示技术及其改进[J].动物医学进展,2004,25(6)：19-21
[63]刘永军,景建洲,贾敬芬,等.用mRNA差异显示技术分离盐胁迫下小麦耐盐相关cDNA[J].西北大学学报,2006,36(1)：89-91
[64]孔凡晶,陈孝,马有志,等.利用差异显示技术克隆小麦抗白粉病相关基因的研究[J].麦类作物学报,2004,24(1)：11-14
[65]黄贵修.利用差异显示技术探索橡胶树死皮病病因的研究[D].华南热带农业大学,1999,16(3)：378-382
[66]陈永华,赵森,严钦泉,等.利用差异显示法研究水稻耐淹涝相关基因[J].农业生物技术学报,2006,14(6)：894-898
[67]湛凤凰.代表性差异分析方法在基因克隆中的应用及其发展[J].国外医学遗传学分册,1998,21(3)：113-116
[68]方孝东,林栖凤,李冠一.对代表性分析方法的改进[J].海南大学学报自然科学版,2003,21(2)：135-138
[69]杨 林,袁爱力,李勇,等.一种基因克隆的新策略一代表性差异分析法[J].国外医学遗传学分册,1998,21(3)：113-116
[70]Diatchenko L, Lau Y F, Campbell A P, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes [J]. Proc Natl Acad Sci USA,1996,93:6025-6030
[71]赵永贞,蒋伟明,陈秀荔,等.抑制性消减杂交技术在对虾基因克隆中的应用[J].动物医学进展,2008,29(4)：76-80
[72]李 茂,字学娟,蒋昌顺.抑制消减杂交在热带作物研究中的应用[J].亚热带农业研究,2008,4(2)：151-153
[73]Money T, Reader S, Qu L J, et al. AFLP-based mRNA fingerprinting [J]. Nucleic Acids Research,1996 (24):2616-2617
[74]Donson J, Fang Y, Espiritu Santo G, et al. Comprehensive gene expression analysis by transcript profiling [J]. Plant Molecular Biology,2002 (48):75-97
[75]李驰,卢新雄,陈晓玲,等.利用cDNA-AFLP技术分析老化棉花种子基因差异表达[J].棉花学报,2007,19(4)：255-260
[76]李凌,宋文芹,毛英伟,等.用cDNA-AFLP银染技术研究与花椰菜花色相关的基因.南开大学学报(自然科学版),2000,33(4)：33-36
[77]刘涛,徐平珍,胡运乾,等.板栗花序与叶的cDNA-AFLP的差异表达分析[J].中国农学通报,2007,23(2)：81-84
[78]余梅,江昌俊,叶爱华,等.利用cDNA-AFLP技术研究茶树花蕾发育基因差异表达片段[J].茶叶科学,2007,27(3)：259-264
[79]Mamati G. E. Tea polyphenols biosynthesis and genes expression control in tea (Camellia sinensis),浙江大学博士学位论文,2005
[80]陈桂平,黄占景,马闻师,等.盐胁迫下小麦耐盐突变体后代差异cDNA的分离和鉴定.中国农业科学,2003,36(9)：996-999
[81]陈桂平,马闻师,黄占景,等.小麦耐盐突变体盐胁迫下SIR73基因片段的分离和鉴定.遗传,2003,25(2)：173-176
[82]张莉,江昌俊,胥振国,等.用cDNA-AFLP技术研究茶树种子在低温贮藏过程中差异基因的表达[J].安徽农业大学学报,2008,35(3)：319-323
[83]吴扬.应用cDNA-AFLP技术分离安吉白茶阶段性返白过程的差异表达基因[D].湖南农业大学硕士学位论文,2009.
[84]丁友成,张辉,钟明安,等.基因芯片技术筛选大肠癌肝转移差异表达基因[J].南京医科大学学报(自然科学版),2008,28(4)：476-479
[85]汤玉瑜,陈永文,费蕾,等.基因芯片筛选HepG2与HepG2.2.15细胞中的差异表达基因[J].免疫学杂志,2008,24(3)：329-333
[86]蒋立坚,赖仁发,李卫国,等.基因芯片筛选成釉细胞瘤差异表达基因的研究[J].中国病理生理杂志,2008,24(5)：992-996
[87]李沛,凌志强,赵继敏,等.建立cDNA微矩阵基因芯片技术并分析食管癌细胞系Eca109基因表达谱[J].生物医学工程学杂志,2008,25(2)：412-417
[88]王冰林,柳俊,李媛媛,等cDNA微阵列鉴定差异表达基因的可靠性分析[J].华北农学报,2008,23(3)：5-8
[89]韩杨云,曾义.生物芯片技术与胶质瘤基因的差异表达[J].国际神经学与神经外科学,2007,34(6)：568-571
[90]徐清华.基因芯片技术的研究进展[J].中国优生与遗传杂志,2007,15(1)：13-14
[91]Deng W W, Ogita S, Ashe K H. Biosynthesis of theanine (γ-ethylamino-1-glutamic acid) in seedlings of Camellia sinensis [J]. Phytochemistry Letters.2008,1(2): 115-119.
[92]杨贤强,王岳飞,陈留记,等.茶多酚化学[M].上海：上海科学技术出版社,2003.
[93]Martens S, Knott J, Seitz CA, et al. Impact of biochemical pre-studies on specific engineering strategies of flavonoid biosynthesis in plant tissues [J]. Biochemical Engineering Journal,2003,14:227-235.
[94]Matsumoto S, Takeuchi A, Hayatsu M, et al. Molecular cloning of phenylalanine ammonium lyase cDNA and classification of varieties and cultivars of tea plants (Camellia sinensis)using a tea PAL cDNA probe [J]. Theor. Appl. Genet.,1994,89: 671-675.
[95]Takeuchi A, Matsumoto S, Hayatsu M. Chalcone synthase from Camellia sinensis: isolation of the cDNA and the organspecific and sugar responsive expression of the genes [J]. Plant Cell Physiol,1994(5):1011-1018.
[96]Takeuchi A, Matsumoto S. Dihydroflavonol 4-reductase mRNA from Camellia sinensis. Http://www.ncbi.Nlm.Nih.Gov,Accessing No.AB018685.
[97]Singh K, Kumar S, Ahuja P S. Cloning and characterization of cDNA encoding transcinnamate 4-hydroxylase from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.AY641731.
[98]Rani A, Kumar S, Ahuja PS. Camellia sinensis 4-Coumaroyl CoA Ligase mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing.No.DQ194356.
[99]Rani A, Kumar S, Ahuja PS. Camellia sinensis Cultivar Upasi-10 Chalcone Isomerasc (CHI) mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No. DQ120521.
[100]Chen L, Ma CL, Zhao LP. Molecular cloning of full-length cDNA of the chalcone isomerase(CHI) gene from Camellia sinensis (L.) O. Kuntze. Http://www.Ncbi.Nlm. Nih.Gov,Accessing No.DQ904329.
[101]Singh K, Kumar S, Ahuja PS.2004 Molecular cloning of full length cDNA of flavanone 3-hydroxylase(f3h) gene from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.Ncbi.Nlm.Nih.Gov,2004,Accessing No.AY641730.
[102]Rani A, Kumar S, Ahuja PS. Camellia sinensis Flavonoid 3',5'-Hydroxylasc mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.DQ194358.
[103]Park J S,Kim J B, Kim Y H. Camellia sinensis leucoanthocyanidin reductase (LCR) mRNA. Http://www.ncbi.Nlm.Nih.Gov,Accessing No.AY169404.
[104]Singh K, Kumar S, Ahuja PS. Cloning of full length cDNA encoding anthocyanidin synthase (ANS) from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www. ncbi.Nlm.Nih.Gov,Accessing No.AY830416.
[105]Singh K, Kumar S, Ahuja PS. Molecular cloning of full length cDNA of leucoanthocyanidin reduetase(LAR) gene from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.ncbi.Nlm.Nih.Gov, Accessing No. AY641729.
[106]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.
[107]Kato M, Mizuno K, Fujimura T, el al. Purification and Characterization of Caffeine Synthase from Tea Leaves [J]. Plant Physiology,1999,120:579-586.
[108]张广辉,梁月荣,吴颖.咖啡碱生物合成研究进展及在茶树育种中的应用[J].茶叶,2005,31(1)：18-23.
[109]吴颖,梁月荣.S-腺苷甲硫氨酸在茶树生理代谢中的研究现状[J].茶叶,2005,31(2)：85-87.
[110]冯艳飞,梁月荣,茶树S-腺苷甲硫氨酸合成酶基因的克隆和序列分析[J].茶叶科学,2001,21(1)：21-25
[111]Kato M. Biochemistry and molecular biology in caffeine biosynthesis-Molecular cloning and gene expressing of caffeine synthase. In:Proceedings of 2001 International Conference on O-Cha (Tea) Science (SessinⅡ),21-24, Shizuoka, Japan.
[112]Mizutani M, Nakanishi H, Ema J, et al. Cloning of β-primeverosidase from tea Leaves, a key enzyme in tea aroma formation [J]. Plant Physiology,2002,130: 2164-2176.
[113]Jiang CJ, Yang SL, Li YI-I, el al. Camellia sinensis beta-1,3-glucosidase-like mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.AF537127.
[114]李远华,江昌俊,余有本.茶树p-葡萄糖苷酶基因mRNA的表达[J].南京农业大学学报,2005,28(2)：103-106.
[115]Rawat R, Gulati A, Hallan V, el al. Molecular characterization of beta-glucosidase from regional Kangra tea clone (Camellia sinensis (L.)O Kuntze). AM285295.
[116]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.
[117]王朝霞,江昌俊,蔡海云.茶树p-1,3-葡聚糖酶基因cDNA片段的克隆与序列分析[J].安徽教育学院学报,2006,24(3)：67-70.
[118]Singh K, Kumar S, Ahuja PS. Camellia sinensis HSP70 (hsp70) mRNA (partial cd). Http://www. Ncbi. Nlm. Nih. Gov, Accessing No. AY694190.
[119]Singh K, Kumar S, Ahuja PS. Cloning and sequencing of catalase gene from Camellia sinensis (L.) O. Kuntze cv. UPASI-10. Http://www.Ncbi.Nlm.Nih.Gov, Accessing No.AY641732.
[120]Singh K, Vyas D, Kumar S, el al. Cloning of cDNA encoding manganese superoxide dismutase (MnSOD) from Camellia sinensis (L.) O. Kuntze. Http:www. Ncbi.Nlm.Nih.Gov,Accessing No.AY641734.
[121]Singh K, Kumar S, Ahuja PS. Camellia sinensis Cu/Zn superoxide dismutase mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.AY694187.
[122]Tomimoto Y, Ikehashi H, Kakeda K, el al. A pistil-specific PR-1 like protein of Camellia, its expression, sequence and genealogical position [J]. Breeding Sci,1999, 49:97-104.
[123]Paul A, Kumar S, Ahuja PS. Camellia sinensis metallothionin 1 mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.DQ442270.
[124]Paul A, Kumnr S, Ahuja PS. Camellia sinensis metallothionin 2 mRNA. Http://www.ncbi.him.nih.Gov,Accessing No.DQ442271.
[125]Paul A, Kumar S, Ahuja PS. Camellia sinensis ascorbate peroxidase mRNA. Http://www.Ncbi.Nlm.Nih.Gov,Accessing No.DQ442272.
[126]Chen L, Zhang YL, Zhao LP. Molecular cloning of full-length cDNA of the ACC oxidase gene from Camellia sinensis (L.) O. Kuntze. Http://www.Ncbi.Nlm.Nih. Gov, Accessing No.DQ904328.
[127]Frohman M A, Dush M K, Martin G R. Rapid production of full-length cDNAs from rare transcripts:Amplification using a single gene-specific oligonucleotide primer [J]. Proc Natl Acad Sci USA,1988,85:8998-9002.
[128]王关林,方宏筠.植物基因工程原理与技术[M].北京：科学出版社,1998158-161.
[129]赵炜,郑佐华,毛裕民.全长cDNA的获得RACE及其他方法进展[J].生命科学,1999,11(2)：92-96.
[130]明洪,黄秉仁RACE-cDNA末端快速扩增技术进展[J].生物工程进展,1997,17(5)：7-12.
[131]张庆华,茅矛,陈竺.基因组研究中全长cDNA克隆的策略[J].生物工程进展,2000,20(4)：3-5.
[132]孔凡晶,马有志,陈孝,等.差异显示和RACE技术的发展与应用[J].生物技术通报,2003,1(4)：13-16.
[133]陈启龙RACE技术的研究进展及其应用[J].黄山学院学报,2006,8(3)：95-98.
[134]LIU X W, GOROVSKY M A. Mapping the 5'and 3'ends of Tetrahymena thermophila mRNA using RNA ligase mediated amplification of cDNA ends(RLM-RACE)[J]. Nucleic Acids Res,1993,21(21):4954-4960.
[135]Maruyama K, Sugano S. Oligo-Capping—A simple method to replace the CAP structure of Eukaryotic messeger-RNAs with oligoribonucleotides [J]. Gene,1994, 138:171-174.
[136]周维一,黄翠琴,王寿昆,等.RACE一种研究新基因的有效方法[J].热带医学杂志,2007,7(4)：386-389.
[137]SCHAEFER BC. Revolutions in rapid amplification of cDNA ends:new strategies for polymerase chain reaction cloning of full-length cDNA ends [J]. Anal Biochem, 1995,227(2):255-273.
[138]Ye Z, Connor JR. cDNA cloning by amplification of circularized first strand cDNAs reveals non-IRE-regulated iron-responsive mRNAs [J]. Biochem Biophys Res Commun,2000,275(1):223-227.
[139]邓雪柯,殷建华,曹毅.3种5'RACE技术的比较与优化[J].成都医学院学报,2007,2(1)：20-25.
[140]董志敏,李英慧,张宝石,等.一种获得大片段克隆的SMART全长cDNA文库构建方法[J].大豆科学,2007,12(3)：223-226.
[141]汤皴.茶氨酸的合成药理及其在食品中的应用.茶叶通报,2002,24(4)：19-21.
[142]陈宗懋.茶氨酸具有降压功能.中国茶叶,1997,2：27.
[143]丁王,丁雪.茶氨酸的抗疲劳作用研究.中国公共卫生,2002,18(3)：315-317.
[144]谭和平,陈丽,叶善蓉,等.茶叶中氨基酸的检测方法概述[J].中国检测技术,2007,33(6)：1-4.
[145]Liepman AH, Olsen LJ. Peroxisomal alanine:glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana [J]. Plant J,2001;25:487-498.
[146]Daisuke Igarashi, Hiroko Tsuchida, Mitsue Miyao, et al. Glutamate:Glyoxylate Aminotransferase Modulates Amino Acid Content during Photorespiration [J]. Plant Physiol,2006,142:901-910
[147]刘丽娜,刘祥林,陈志玲.细胞周期调控的一个重要元素-CDC48[J].生物技术通报,2006,5：26-29
[148]Frank Madeo, Jan Schlauer, Hans Zischka, et al. Tyrosine Phosphorylation Regulates Cell Cycle-dependent Nuclear Localization of Cdc48p[J]. Molecular Biology of the Cell,1998,9(1):131-141