茶树茶氨酸合成酶基因的酶活性验证与蛋白三维结构分析

针对NCBI上已登录的茶氨酸合成酶与谷氨酰胺合成酶基因序列进行克隆、原核表达与酶活性验证,利用多种生物信息学数据库和软件,对Cs TS与Cs GS基因进行结构、性质和功能预测,采用同源建模法对蛋白三维结构进行预测,比较并预测催化作用位点的差异;用系统进化树分析从裸子植物到高等被子植物的谷氨酰胺合成酶基因序列,推测其进化的演变过程;通过对原核表达的基因工程菌提取粗酶液进行酶活性测定。结果表明:尽管茶树TS与GS序列高度同源,但是原核表达后的融合蛋白仍然显示了不同的催化能力,蛋白一、二级结构分析显示Cs TS与Cs GS差异不大,但是通过同源建模形成的蛋白三级结构分析显示,Cs TS与Cs GS存在3个催化位点上的差异,这可能是导致其酶活性差异的关键。系统进化分析结果首次确定茶氨酸合成酶应为谷氨酰胺合成酶基因家族成员,按照其细胞定位预测应为胞质型GS,其亲缘关系与同为双子叶植物的葡萄、陆地棉、巴西橡胶树、拟南芥较接近。     

夏季遮阴对茶树茶氨酸合成及其代谢相关基因表达的影响（英文）

为了研究夏季遮阴对茶树茶氨酸代谢途径的影响及对茶树内含物品质的改良作用,本研究以多年实生茶树为研究对象,利用Western blotting检测了夏季遮阴对茶树不同部位中茶氨酸合成酶(TS)、谷氨酰胺合成酶(GS)蛋白表达的影响,确定遮阴有利于嫩叶中TS的表达;随后利用实时荧光定量PCR检测方法探索了茶氨酸代谢途径中相关基因:TS、GS、谷氨酰胺α-酮戊二酸氨基转移酶(GOGAT)、谷氨酸脱氢酶(GDH)以及与氮素吸收和转化密切相关的亚硝酸还原酶(NiR)、精氨酸脱羧酶(ADC),在老、嫩叶中及不同遮阴时期的表达情况。结果表明遮阴有利于嫩叶中TS基因的表达,与叶部氨的再同化作用密切相关的GS、GOGAT和GDH基因均在老叶中有明显增加,而与硝基氮代谢相关的NiR、ADC基因表达在老叶与嫩叶中均明显下降。利用HPLC对遮阴后嫩叶中游离氨基酸含量的检测结果表明,遮阴明显促进茶叶部游离氨基酸总量的提高,其中贡献率最大是茶氨酸。本研究从分子水平上解析了遮阴促进茶叶部茶氨酸积累的调控机制,为今后夏秋茶栽培措施改良和品质改善提供理论基础。     

茶树谷氨酰胺合成酶同源基因的克隆及表达分析

在实验室前期构建cDNA幼根文库获得谷氨酰胺合成酶(glutamine synthetase,GS,EC 6.3.1.2)同源序列(contig48)的基础上设计引物,通过SMART RACE技术克隆了该基因cDNA全长序列(命名为GS1-2,GenBank登录号:JQ925873.1)。结果显示:(1)GS1-2基因全长为1 710bp,开放阅读框长1 071bp,编码356个氨基酸,预测蛋白分子质量为39.3kD,理论等电点为5.65;核酸序列分析表明,GS1-2基因与从安吉白茶中克隆的茶氨酸合成酶基因相似性为99%。(2)将GS1-2基因克隆至原核表达载体pET-32a和pMAL-c5x,转化至大肠杆菌,IPTG诱导表达融合蛋白,SDS-PAGE检测结果表明,pET-32a-CsGS1-2转至Rosetta中诱导表达的蛋白与预测蛋白大小一致,主要以包涵体形式存在;而pMAL-c5x-CsGS1-2转化大肠杆菌BL21(DE3)诱导表达可产生可溶性蛋白。(3)进一步构建茶树GS1-2酵母表达载体pYES-DEST52-CsGS1-2并转化至酿酒酵母(WAT11)菌液中,添加底物(谷氨酸钠100μmol/L和盐酸乙胺500μmol/L)震荡培养并离心,UPLC-MS测定酶反应产物结果初步表明,目的蛋白不能催化盐酸乙胺和谷氨酸钠合成茶氨酸,但可以合成谷氨酰胺。     

铁观音原生质体高效瞬时转化方法的建立

近年来, 茶树基因组测序的完成为茶树在分子和基因水平的研究奠定了基础。但由于转基因技术尚不成熟且茶树生长周期较长, 茶树的基因功能研究依然不能有效开展。采用铁观音(Camellia sinensis var. sinensis cv. ‘Tieguanyin’)实生幼苗叶片, 通过筛选多种纤维素酶、果胶酶、离析酶和甘露醇的浓度组合, 并结合原生质体的数量、活性和杂质含量综合确定了最佳配方, 成功建立了铁观音茶苗叶片原生质体提取和PEG介导的高效瞬时转化体系, 转化率达56.25%。利用该系统探索了茶氨酸代谢通路中2个重要合成酶(茶氨酸合成酶(TSI)和谷氨酰胺合成酶(GSII-1.1))的亚细胞定位。研究发现, 这2种酶均定位于铁观音原生质体细胞质中。茶苗叶片原生质体提取和瞬时转化体系的建立为茶树基因组功能研究奠定了技术基础。     

茶氨酸的制取及应用

茶氨酸是茶叶中的一种主要氨基酸 ,通常占茶叶干重的 2 %左右 ,约占茶树体内游离氨基酸5 0 %。茶氨酸具有鲜甜味 ,是茶叶特征物质之一 ,与茶叶品质呈正相关。现已作为食品添加剂应用于食品领域。除此以外 ,茶氨酸还具有一些重要的药理作用。如抗肿瘤、降压安神、拮抗咖啡碱等医疗功效。本文综述了茶氨酸的性质、制取方法及应用前景等方面的内容。     

野生大豆根瘤GmGS1γ基因序列分析及原核表达

旨在明确大豆谷氨酰胺合成酶(Glutamine synthetase,GS)基因家族各成员的结构特点及功能。利用同源克隆的方法从野生大豆根瘤克隆Gm GS1γ基因。生物信息学分析表明,ORF为1 071 bp,与大豆GS1γ(AF363022.1)部分序列的相似性为100%,与序列号为X81700.1相似性为99%。该序列具备植物GS的两个保守结构域,GS beta-Grasp功能区(17-97 aa)和GS催化功能区(103-350 aa)。系统发生树表明该基因编码的GS可能属于胞质2型同工酶。该基因可以在大肠杆菌DE3.0中表达,蛋白分子量为44 k D。     

茶氨酸和没食子酸在普洱茶中的含量变化

建立高效液相色谱分析茶氨酸和没食子酸的方法,对由云南大叶茶（Camellia sinensis var．assamica）生产的晒青毛茶及其加工的普洱茶中二者的含量进行分析。结果表明,普洱茶中没食子酸的含量显著增高,而茶氨酸的含量则明显降低。茶氨酸和没食子酸的含量与原料的来源和质量,以及普洱茶的后发酵生产过程均有关系。对二者含量变化的机制进行了初步的讨论。     

纸层析－分光光度法检测茶氨酸

为了满足普通实验室对茶中茶氨酸测定的需要,研究了茶氨酸的纸层析-分光光度检测方法。结果表明,用茚三酮．乙醇水溶液做展层剂,对茶苗根、芽叶和茶叶的水浸提液进行纸层析,能够有效地将茶氨酸与其它氨基酸分离,紫色色斑清晰而均匀。用乙醇溶液洗脱色斑后用分光光度计在570nm比色,在20～70此茶氨酸溶液点样量范围内其含量与吸光度呈线性关系。本方法检出限为0．0057mg·mL-1,测定下限为0．0191mg·mL-1,平均回收率90．28％～115．38％,平均相对标准差1．51％,具有安全、药品种类少和操作步骤简单等特点。     

茶氨酸保健功能研究进展

茶氨酸是茶叶中特有的氨基酸,是茶叶重要活性成分之一。国内外大量研究表明它在保护神经、镇静、调节情绪、提高认知能力等方面有良好的保健作用。最新的研究进展表明茶氨酸可以通过激活T淋巴细胞起到抗肿瘤的作用。作为保健品,茶氨酸在医药和食品加工方面也有着良好的应用前景。     

沉淀法从茶叶中提取茶氨酸

茶叶浸提液分别用1%壳聚糖和D101大孔吸附树脂去杂后,用碱式碳酸铜沉淀茶氨酸。茶氨酸铜盐用1mol/L硫酸解析,分别用H_2S、Ba(OH)_2除去Cu~(2 )、SO_4~(2 )后,浓缩重结晶得到茶氨酸,提取率34%,纯度99．28%。     

大规模茶叶细胞悬浮培养茶氨酸合成工艺优化研究

以婺源绿茶为材料进行茶叶愈伤组织悬浮培养,采用正交实验设计进行了大规模茶叶细胞悬浮培养合成茶氨酸工艺条件优化研究。结果显示,NH4+/NO-30.0/60.0mmol/L、K+100.0mmol/L、Mg++3.0mmol/L、H2PO-43.0mmol/L、蔗糖30.0g/L、水解酪蛋白2.0g/L条件下,茶叶细胞生长量(速率)和茶氨酸积累量均达到最高值;提高培养基中蔗糖和水解酪蛋白浓度可延长细胞对数生长期和稳定生长期,从而有利于茶氨酸积累;H2PO-4浓度主要影响细胞生长速率和茶氨酸积累速率的同步性,低H2PO4-浓度环境中茶氨酸积累速率峰值滞后于细胞增长速率峰值,高H2PO4-浓度环境中早于细胞生长速率峰值出现时间;K+和Mg++对细胞生长的影响不明显,但影响茶氨酸合成酶活性,维持适量的K+和Mg++有利于茶氨酸积累。先加入一定量盐酸乙胺再每天进行少量补充,茶氨酸合成量比一次性加入的效果要好。从生产效率考虑,培养周期以19～22d为宜。     

Pharmacokinetics of theanine enantiomers in rats

Theanine, first discovered in tea, is a chiral nonproteinic amino acid that has been reported to have cardiovascular, neurological, and oncological effects. It is being considered as a therapeutic/medicinal agent and additive in consumer products. The present study evaluated the pharmacokinetics of D-theanine, L-theanine, and D,L-theanine in plasma and urine using LC-ESI/MS in rats after oral (p.o.) and intraperitoneal (i.p.) administration. Oral administration data indicated that gut absorption of d-theanine was far less than that of L-theanine. However, after i.p. administration, plasma theanine concentrations of L- and D-theanine were similar. This indicated that D- and L-theanine may exhibit a competitive effect with respect to intestinal absorption. Regardless of the route of administration, p.o. or i.p., the presence of the other enantiomer always decreased theanine plasma concentrations, indicating D,L-theanine competition with respect to urinary reabsorption. Data on urinary concentrations of D-theanine suggested that the D-isomer may be eliminated with minimal metabolism. L-Theanine appeared to be preferentially reabsorbed and metabolized by the kidney while D-theanine was preferentially excreted. Clearly, the bioequivalencies of D,L-theanine and its enantiomers were found to be quite different from one another. Consequently, the efficacy of commercial theanine products containing D-theanine, L-theanine, or D,L-theanine may be quite different.     

Inhibitory effects of theanine and sera from theanine-fed rats on receptor-mediated cancer cell invasion beneath mesothelial-cell monolayers

To investigate the bioavailability and mode of action of theanine against cancer, we examined in vitro and ex vivo effects of theanine on invasion of a rat ascites hepatoma cell line of AH109A. Theanine dose-dependently inhibited the invasion of AH109A cells across rat mesentery-derived mesothelial-cell (M-cell) monolayers without restraining AH109A cell proliferation in vitro. Rat sera obtained after oral intubation of theanine also inhibited the invasion. A competitive N-methyl-D-aspartate (NMDA) type glutamate receptor antagonist, (±) 2-amino-5-phosphonopentanoic acid (AP-5), dose-dependently counteracted the theanine-mediated in vitro and ex vivo inhibition of AH109A invasion. A competitive non-NMDA type glutamate receptor antagonist, 6,7-dinitroquinoxaline 2,3-dione (DNQX), did not affect this inhibition by theanine in vitro. These results suggest that the inhibition of AH109A invasion by theanine may be mediated by the NMDA receptor of AH109A. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nonaqueous fractionation and overexpression of fluorescent‐tagged enzymes reveals the subcellular sites of L‐theanine biosynthesis in tea

l ‐Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of l ‐theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing l ‐theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution of l ‐theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of l ‐theanine synthetase and also to determine the subcellular localization of fluorescent‐tagged proteins by confocal laser scanning microscopy. In tea root tissue, the cytosol was the main site of l ‐theanine biosynthesis, and cytosol‐located CsTSI was the key l ‐theanine synthase. In tea shoot tissue, l ‐theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key l ‐theanine synthases. In addition, l ‐theanine content and distribution were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.     

茶氨酸的分析测试研究

综述了近年来茶氨酸的分析测试方法,对各种方法的优缺点进行了比较,并对未来的发展方向做了展望。     

Distribution and biosynthesis of theanine in Theaceae plants

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 μmol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [¹⁴C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

ZtNH2-HCl和剪切力对茶叶细胞悬浮培养中茶氨酸合成的影响

以婺源绿茶嫩叶愈伤组织为材料,在采用摇床悬浮培养与发酵罐悬浮培养．分析了茶氨酸合成前体盐酸乙胺(ZtNH2-HCl)不同的添加方式和剪切力对培养细胞增长量和茶氨酸合成量的影响。结果显示,发酵罐放大培养取得了与摇床悬浮培养类似的效果;在一个培养周期中,培养细胞茶氨酸积累高峰出现在第20～22天;发酵罐大规模培养时采用桨叶式搅拌器(低剪切力)细胞增长量和茶氨酸合成苗优于标准板搅拌器;添加盐酸乙胺可大幅度提高茶氨酸积累量,先加入一定量盐酸乙胺再每天进行少量补充,茶氨酸合成最比一次性加入的效果要好。