不同培养条件对悬浮培养茶叶细胞生长及茶氨酸合成的影响*

婺源绿茶嫩叶用MS 培养基( 加IBA 2 mgPL, 6-BA 4 mgPL) 进行茶叶愈伤组织悬浮培养, 研究了不同培养条件对茶叶细胞悬浮培养过程中细胞生长与茶氨酸合成的影响。结果显示, NH4+PNO3- 110P6010 mmolPL、K+ 10010 mmolPL、Mg2+ 310mmolPL、H2PO4- 310 mmolPL、蔗糖3010 gPL、水解酪蛋白210 gPL 条件下, 茶叶细胞生长量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使细胞对数生长期和稳定期得到延长, 从而有利于茶氨酸积累; H2 PO4- 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H2 PO4- 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Mg2+ 对细胞生长的影响不明显, 但影响细胞茶氨酸合成酶活性, 维持适量的K+和Mg2+ 有利于茶氨酸积累。添加盐酸乙胺可大幅度提高茶氨酸积累量, 并且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。茶叶细胞生长和茶氨酸积累高峰期在整个培养过程的第19~ 22 天出现, 从生产效率考虑, 培养周期以19~ 22 天为宜。     

不同培养条件对悬浮培养茶叶细胞生长及茶氨酸合成的影响

婺源绿茶嫩叶用MS培养基（加IBA 2mg／L,6-BA 4mg／L）进行茶叶愈伤组织悬浮培养,研究了不同培养条件对茶叶细胞悬浮培养过程中细胞生长与茶氨酸合成的影响。结果显示,NH4^＋／NO3^- 1．0／60．0mmol／L、K^＋ 100．0mmol／L、Mg^2＋ 3．0mmol／L、H2PO4^- 3．0mmol／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使细胞对数生长期和稳定期得到延长,从而有利于茶氮酸积累;H2PO4^-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^2＋对细胞生长的影响不明显,但影响细胞茶氨酸合成酶活性,维持适量的K^＋和Mg^2＋有利于茶氨酸积累。添加盐酸乙胺可大幅度提高茶氨酸积累量,并且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。茶叶细胞生长和茶氨酸积累高峰期在整个培养过程的第19～22天出现,从生产效率考虑,培养周期以19～22天为宜。     

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。     

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。     

藏红花细胞悬浮培养体系的建立及优化

基于诱导的藏红花细胞系,通过摇瓶法,优化了其液体培养基、接种量和种龄等培养条件,以建立藏红花细胞悬浮培养体系。结果表明,将生长在固体培养基上的藏红花愈伤组织接种在MS液体培养基(添加了2mg/L2,4-D,1mg/L6-BA和300mg/LCH)中,于(22±0.3)℃,120r/min的摇床上,暗培养30d,便可获得藏红花的悬浮细胞系。经优化其培养基、接种量和种龄,将种龄为20d的细胞系,按照5%接种量接种在液体B5培养基(添加了2mg/LNAA,1mg/L6-BA和300mg/LCH)中,于(22±0.3)℃,120r/min的摇床上,培养36d,细胞生物量(13.4g/L)和藏红花素产量(0.91g/L)均达到最高。本研究建立的藏红花细胞悬浮培养体系为其生物反应器放大培养奠定了基础。     

茶条槭悬浮培养体系的建立与没食子酸合成的优化条件

初步建立茶条槭(Acer ginnala)细胞悬浮培养体系: 以茶条槭子叶为外植体, 接种于WPM培养基中, 对茶条槭愈伤组织进行诱导和继代培养。悬浮培养中, 每代增长指数达到11.6, 没食子酸含量达到1.518%。通过对比NT、IS、WPM、B5和MS培养基所含成分对茶条槭愈伤组织悬浮培养的影响, 综合考虑悬浮细胞的生长速率和有效成分的含量, 确定WPM为基本培养基。WPM培养基大量元素的浓度对细胞的生长和没食子酸的积累有显著影响, 其浓度越高, 促进作用越明显。3倍浓度的大量元素最有利于没食子酸的积累。蔗糖浓度为10 g.L-1最适于没食子酸的积累, 浓度为30 g.L-1最适于茶条槭细胞生长和没食子酸合成。     

紫花曼陀罗悬浮培养细胞转化对羟基苯甲醛生产天麻素

利用紫花曼陀罗细胞悬浮培养转化外源对羟基苯甲醛合成天麻素,并应用多种色谱技术进行分离纯化,根据转化产物的理化性质和光谱数据分析鉴定结构。实验表明,紫花曼陀罗细胞成功将对羟基苯甲醛转化为天麻素(Ⅱ),同时也得到了由对羟基苯甲醛生成天麻素的转化中间体对羟基苯甲醇(Ⅰ)。在培养基中添加0.1mg/L的水杨酸能显著提高细胞对外源对羟基苯甲醛的糖基化率,而保持气升式发酵罐(25-L)罐内压力为低压(0.001MPa)也能提高细胞对外源对羟基苯甲醛的糖基化率。实验证明,紫花曼陀罗细胞悬浮培养能有效转化对羟基苯甲醛合成天麻素。     

水母雪莲悬浮培养细胞生长和黄酮类活生成分合成

在ＭＳ培养基上进行水母雪莲细胞悬浮培养,研究了摇床转速、接种量、培养液初始ｐＨ、碳源等的影响。结果表明,摇床转速为９０￣１２０ｒ／ｍｉｎ,接种量为５０￣８０ｇＦＷ／Ｌ,培养液初始ｐＨ５．５￣６．０,对水母雪莲悬浮培养细胞生长和黄酮合成最有利。碳源以蔗糖最适合,蔗糖浓度则以４０ｇ／Ｌ较好,此时细胞生长量为１８￣１９ｇＤＷ／Ｌ,总黄酮合成量可达１４２３．２５ｍｇ／Ｌ。用ＨＰＬＣ检测显示４＇,５,７－三     

利用固定化假单孢菌细胞生产茶氨酸的新方法

L-茶氨酸可增加冲饮绿茶的品味,最初发现于茶树的根尖,通常采用化学合成。曾有报道经茶氨酸合成酶作用由谷氨酸和乙胺合成,唯因产率低及     

内生真菌对油樟悬浮细胞培养的影响

该文研究了内生真菌YG42、YG71、YY11和YY26发酵液,对油樟悬浮细胞的生长量及挥发性代谢产物积累量的影响。结果表明：4种内生真菌对油樟悬浮细胞的生长均有抑制作用,抑制强度随发酵液添加量的增加而加强。4种内生真菌对油樟悬浮细胞挥发性代谢产物积累总量及1,8-桉叶油素、γ-叶松油烯和α-油松油醇3种油樟油组分物质积累量的影响多表现为低浓度促进高浓度抑制的趋势。其中,1％添加量的YG42和YY26及0.25％添加量的YY11对悬浮细胞总挥发性代谢产物积累的促进作用相当且最强,其积累量分别是空白组的2.00、1.95、2.01倍;0.25％添加量的YG71对1,8-桉叶油素积累的促进作用最强,其积累量为空白组的11.03倍;0.25％添加量的YG71和YY26对α-松油醇积累的促进作用相当且最强,其积累量分别为空白组的1.72和1.81倍;对于γ-松油烯的积累,在空白组中未检测到其峰值,4种真菌诱导子对γ-松油烯的产生有诱导作用,诱导的最大峰面积为0.19,诱导菌是0.25％添加量的YG71。该研究结果为充实内生菌影响香料植物挥发性代谢产物合成理论奠定了基础,也为生产上内生真菌提高油樟油中有用物质组分含量措施的采用提供了依据。     

弧菌菌株发酵工艺初步研究

采用摇瓶培养和发酵罐培养并在不同培养时间测定菌液浊度和活菌数,为弧菌灭活疫苗生产工艺提供参数.研究结果表明,摇瓶培养装量以30%较为合适,培养基初始pH 7.5,发酵过程以不再控制为宜.初步确定了弧菌发酵培养工艺参数:弧菌菌株接种TSB,28 ℃摇瓶培养18～20 h,28 ℃种子罐通气搅拌培养12～14 h,28 ℃...     

Some Properties of the Theanine Synthesizing Enzyme in Tea Seedlings

Characterization of the theanine synthesizing enzyme found in tea seedlings was carried out. Evidences suggest that this enzyme seems to be a synthetase peculiar to the tea plant, having a high affinity for ethylamine.     

Theanine transporters identified in tea plants (Camellia sinensis L.)

Theanine, a unique non‐proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H⁺‐dependent. The tissue‐specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root‐to‐bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root‐to‐shoot delivery in the tea plant.     

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。     

中国仓鼠卵巢细胞的悬浮适应及代谢特征

目的：通过悬浮适应,使中国仓鼠卵巢细胞（CHO细胞）获得悬浮生长的特性,并可在悬浮培养条件下较快地生长。方法：将CHO细胞以3×10^5／mL接种于100mL的三角瓶内,培养时加入1％小牛血清、1g/LPIuronic F-68、25μg／mL硫酸葡聚糖,培养体积35mL,摇床转速90r／min,每24h离心换液,当细胞增殖为2×10^6／mL时传代。结果：经过悬浮适应,细胞的平均比生长速率由适应最初的0．27／d提高为适应后的0．48／d,最大总细胞密度由适应初期的2．5×10^6／mL提高为适应后的6．3×10^6／mL,目的蛋白活性也由适应前的2781U／mL提高为适应后的8878U／mL,适应后细胞的葡萄糖平均比消耗率为1．42μmol／（10^6细胞·d）,低于适应前的2．16μmol／（10^6细胞·d）。结论：贴壁生长的CHO细胞经过悬浮适应,不仅可以在悬浮培养条件下快速生长,而且细胞对葡萄糖的利用率也得到提高。     

l-Alanine as a precursor of ethylamine in Camellia sinensis

After absorption of ammonium nitrogen, nitrogen-deficient Camellia sinensis synthesized theanine following synthesis of glutamic acid and alanine. The rate of incorporation of ¹⁴C from l-alanine U-¹⁴C into theanine was faster than from acetaldehyde 1–2¹⁴4C. Incorporation of ¹⁴C from l-alanine U-¹⁴C into the ethylamide of theanine was prevented by adding an excess of ethylamine to the culture solution. Green seedlings converted alanine to ethylamine more rapidly than did etiolated seedlings.     

Photoautotrophic growth of cell suspension cultures fromChenopodium rubrum in an airlift fermenter

Summary This communication describes the construction and operation of an airlift fermenter for the photoautotrophic growth of cell suspension cultures fromChenopodium rubrum. The basic batch culture unit provides a culture of 1.51 volume, sufficient to permit frequent aseptic sampling. It can be maintained at any desired temperature and aerated to different extents. Using an initial cell density of about 400,000 cells per ml suspension, the increase in cell number is 270% after a 14 days' growth period, although the stationary phase of growth is not yet reached. The transfer of photoautotrophic cell suspensions fromChenopodium rubrum from stationary growth into the large volume of fresh culture medium in the airlift fermenter results in an immediate protein formation, followed by an exponential phase of cell division, whereas rapid chlorophyll accumulation is delayed by 2 days.The growth capacities of photoautotrophic fermenter cultures including protein and chlorophyll formation as well asin vitro activities of the ribulosebisphosphate carboxylase and the phosphoenolpyruvate carboxylase are greatly lower as compared to photoautotrophic cells propagated in standard two-tier culture vessels using 30 ml culture medium. However the pattern of change in the activities of carboxylation enzymes is quite similar in both culture systems.Photoautotrophic cell suspensions fromChenopodium rubrum grown in an airlift fermenter assimilate about 90 mol CO₂/mg chlorophyll × hour. Dark CO₂ fixation is about 1.5% of the light values.Abbreviations PEF phosphoenolpyruvate - RuDP ribulosebisphosPhate - NS ground glass joints of standardized size made from Duran glass, Schott, Germany     

Unique Induction of CA1 LTP Components After Intake of Theanine, an Amino Acid in Tea Leaves and its Effect on Stress Response

Theanine, γ-glutamylethylamide, is one of the major amino acid components in green tea. This study was undertaken to evaluate the effect of theanine intake on long-term potentiation (LTP) induction at hippocampal CA1 synapses and exposure to acute stress. Young rats were fed water containing 0.3% theanine after birth. Key findings: Serum corticosterone level was markedly decreased by theanine intake. Because this decrease can modify synaptic plasticity, the effect of theanine intake was examined focused on CA1 LTP induction. CA1 LTP induced by a 100-Hz tetanus for 1 s was almost the same extent in hippocampal slices from theanine-administered rats, whereas that induced by a 200-Hz tetanus for 1 s was significantly attenuated. 2-Amino-5-phosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly attenuated CA1 LTP induced by a 200-Hz tetanus in the control rats, but not in theanine-administered rats. Interestingly, APV completely blocked CA1 LTP induced by a 100-Hz tetanus in the control rats, while scarcely blocking it in theanine-administered rats. These results indicate that theanine intake reduces NMDA receptor-dependent CA1 LTP, while increasing NMDA receptor-independent CA1 LTP. Furthermore, neither 100-Hz tetanus-induced LTP nor 200-Hz tetanus-induced LTP was attenuated in theanine-administered rats after exposure to tail suspension stress, suggesting that the lack of NMDA receptor-dependent CA1 LTP by theanine intake is involved in ameliorating the attenuation of CA1 LTP after tail suspension. This study is the first to indicate that theanine intake modifies the mechanism of CA1 LTP induction.