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。     

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。     

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。     

提高狗蔷薇离体培养植株再生频率*

以狗蔷薇( Rosa canina Inermis) 为材料, 以MS 为基本培养基, 通过对不同植物生长调节剂的组合,
大幅度提高了狗蔷薇离体培养植株再生频率。结果表明, 210 mgPL 6-BA+ 01 3 mgPL 2, 4-D 的组合较为适宜,
其不定芽再生率为87%, 增殖率为310; 而CPPU 和2, 4-D 的适宜组合为115 mgPL+ 013 mgPL, 其不定芽再
生率高达93%, 增殖率为510。同时, 研究结果显示, 以MS+ 40 gPL 蔗糖+ 610 gPL 琼脂粉+ 31 5 mgPL
AgNO3 + 115 mgPL CPPU+ 01 1 mgPL 2, 4-D+ 0105 mgPL GA3 作增殖培养基效果最好, 不定芽诱导率为89%,
增殖率为51 5; 利于生根的培养基为1P4MS+ 20 gPL 蔗糖+ 315 gPL 琼脂+ 013%活性碳+ 011 mgPL IBA+ 011
mgPL NAA, 生根率为91%。     

不同培养条件对半夏悬浮培养细胞生长及总生物碱形成的影响

研究了半夏悬浮培养过程中细胞鲜重的变化和MS培养基组分中碳源、钙盐、Fe盐及肌醇变化对半夏悬浮培养细胞生长及总生物碱合成的影响。结果表明 ,半夏细胞的生长曲线呈“S”型 ,细胞的最佳收获时间为 2 1d。在两种碳源中 ,葡萄糖比蔗糖利于细胞的生长和总生物碱的合成 ,最适浓度为 2 0g L。最适合细胞生长和总生物碱形成的钙盐浓度为 1 8mmol L、Fe盐浓度为 0 0 6mmol L和肌醇浓度为 10 0mg L。     

主要营养成分对悬浮培养玫瑰茄细胞生长和花青素合成的影响

本文研究了培养基中碳源和氮源变化对悬浮培养玫瑰茄细胞生长和花青素合成的影响。在８种不同的碳源中,麦芽糖有利于花青素的积累,而蔗糖和葡萄糖适合细胞生长,并有较高的花青素产率。在１％～１０％蔗糖浓度范围内,４％浓度下细胞生长和花青素产率最高,而６％浓度下细胞花青素含量最高,高渗环境较有利于细胞花青素的积累。１３５ｍＭ的氮源总量已足够维持玫瑰茄细胞生长和花青素合成,氮源总量增加对细胞代谢有抑制作用。ＮＨ＋４对细胞有显著抑制作用。总量１３５ｍＭ,ＮＯ－３与ＮＨ＋４比例２５∶２和２３∶４时细胞生长和花青素合成最佳。     

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10g·L^-1最适于没食子酸的积累,浓度为30g·L^-1最适于茶条槭细胞生长和没食子酸合成。     

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。     

一株腈水合酶产生菌的培养及酶转化试验

诺卡氏菌KY1023能利用乙腈作为生长的碳源和氮源.最适培养条件为(g·L-1)葡萄糖10,醇母膏20,玉米浆10,诱导剂10,MgSO4·7H2O 0.5,K2HPO40.5,KH2PO40.5,pH7.0,菌株在28℃、250rpm条件下培养24h,丙烯酰胺酶活力达到1330μ/ml.休眠细胞在3h以内,可积累丙烯酰胺浓度达到250g·L-1,乙酰胺浓度达到400g·L-1.     

人参寡糖素M对红花培养细胞生长与α-生育酚形成的影响

人参寡糖素M能提高红花(Carthamus tinctorius)培养细胞的生长速率和培养细胞中代谢产物α-生育酚的含量。其最适作用浓度在愈伤组织培养中为5mg/L。在红花细胞悬浮培养加入人参寡糖素M1d后,培养细胞中α-生育酚的含量即提高,但由于累积效应,因而于细胞接种当天同时加入人参寡糖素M对细胞生长和α-生育酚含量的提高效果较好。加入人参寡糖素M可缩短红花悬浮培养细胞生长的延缓期,并于指数生长期作用最明显;另外可使细胞生长及α-生育酚积累同时提前达到最高值,因而缩短了细胞收获时间。     

Hydroxymethylglutaryl Coenzyme A Reductase in Fusarium oxysporum

Both growth and theanine accumulation in tea callus cultures were improved by the combination of 4 mg/liter benzyladenine and 2mg/liter indol-3-butyric acid, but were strongly inhibited by the addition of 2,4-dichlorophenoxyacetic acid. The optimum initial concentration of carbon source was 30g/liter sucrose. Upon the addition of more than 30 g/liter of sucrose, the callus fresh weight was increased, but the theanine formation was not improved.     

培养基组分对沙打旺(Astragalus adsurgens Pall)组培根增殖的影响及其培养滤液提取物的化感活性

采用L9 (315 )正交设计,研究了B5培养基营养组分对沙打旺组培根增殖的影响;并采用玻璃皿滤纸培养法,对其培养滤液提取物进行生物测定以验证沙打旺组培根的化感活性.结果显示:培养基的所有营养组分中,Fe2 对沙打旺组培根增殖的影响最大,蔗糖、H2PO 4、 Mg2 、 Mn2 、 Cu2 、 Zn2 、 BO3-3、 Co2 、 I-、C8H12ClNO3 C12H18Cl2N4OS C6H5O2N C6H12O6的影响次之,氮、Ca2 、MoO2-4 和NAA的影响最小.根据不同养分条件下沙打旺组培根干重的极差分析,筛选出适宜沙打旺组培根快速增殖的优化培养基.培养滤液提取物的生物测定结果表明沙打旺组培根培养过程中可能产生化感物质;化感作用强度的差异预示营养胁迫可能影响其化感物质的产生.研究为沙打旺组培根再生与繁殖提供一定依据,并揭示养分条件可能是该植物表达化感作用的影响因素.     

Isolated Coxiella burnetii synthesizes DNA during acid activation in the absence of host cells

Two populations of Coxiella burnetii were isolated from fibroblast tissue cultures and examined for their ability to synthesize DNA when incubated in a defined medium. Both the populations released by mechanical lysis of heavily infected host cells, as well as those recovered from the tissue culture medium, incorporated H3 32PO4 into DNA. Incorporation occurred at pH 4.5 but not at pH 7.0, and proceeded for 12-15 h. When incorporation of [3H]thymidine was studied, only the organisms obtained by mechanical lysis of host cells were active. Those which had been released by natural means into the tissue culture medium, and then recovered for study, did not incorporate precursor thymidine but were extremely active in protein biosynthesis. In mechanically released organisms, thymidine incorporation was inhibited immediately by rifamycin (40 microM) and hydroxyurea (10 mM), but it was not affected by chloramphenicol (310 microM) until 4 h after addition of the drug. Incorporation of H3 32PO4 by both populations of organisms was also inhibited by rifamycin, chloramphenicol and hydroxyurea, but the time sequence of inhibition differed. Southern hybridization utilizing 32P-labelled DNA suggested that both populations synthesized authentic chromosomal DNA sequences, as well as QpH1 plasmid DNA, during acid activation of metabolism.     

条件培养液对红豆杉细胞Paclitaxel生产的促进作用

在两步法红豆杉（Taxus chinensis)细胞悬浮培养体系的生产阶段,加入从生长阶段悬浮培养物中制得的条件培养液（conditioned Medium,CM)既能促进细胞的生长,又能提高紫杉醇（paclitaxel)的产率,解决了生产培养时,细胞生长受抑制的问题,特别是,取自生长12天的细胞悬浮培养物的CM按体积分数为25％添加到新鲜生产培养基中时,可使细胞紫杉醇最高产量达28．5mg/L,细胞干重达32．3g/L,分别是对照的2．4倍和2．2倍,对CM中的蔗糖,果糖,NO3－和PO4－3等的含量的进行了分析。     

Reversibility of energy-dependent Ca2+ accumulation in mitochondria

Ca2+ accumulation in energized rat liver mitochondria has been studied after the blockage of mitochondrial permeability transition pore (MPTP) by cyclosporin A. It is shown that Ca2+ transport is coupled to the countertransport of protons: from the matrix of mitochondria in the medium in the course of Ca2+ accumulation, and, on the contrary, from the medium to mitochondrial matrix after membrane depolarization. In standard incubation medium containing K+, Cl-, oxidation substrate (glutamate) and inorganic phosphate (H2PO4(-)) the observed stoichiometry of the exchange is 1Ca2+ : 1H+. In accordance with this exchange ratio, proton, as well as cation, transport follows the same first-order kinetics, which is characterized in both cases by very close values of reaction half-times and rate constants. It is shown that reversion of Ca2+ -uniporter, sensitive to ruthenium red, is necessary for Ca2+ - efflux from the matrix ofdeenergized mitochondria when MPTP is blocked by cyclosporin A. It is also shown that Ca2+ -uniporter reversion takes place only after membrane depolarization and permeabilization by protonophore CCCP. Calcium release from mitochondria in the presence of CCCP is accompanied by proton flow into the matrix. Both calcium and proton fluxes are sensitive to Ca2+ uniporter blocker, ruthenium red, which gives the evidence of the identity of Ca2+ -efflux and influx pathways. The data obtained lead to the conclusion that calcium-proton exchange is necessary for Ca2+ -uniporter reversion and the reversibility of energy-dependent Ca2+ -uptake in mitochondria.     

Responses to sucrose and glutamine by soybean embryos grown in vitro

Immature soybean (Glycine max [L.] Merr. cv. Ransom) embryos were grown in vitro in the presence of different concentrations of sucrose and glutamine to examine how availability of carbohydrate and nitrogen affects dry matter accumulation and embryo composition. Embryos were transferred to fresh medium every 4 days to maintain sucrose and glutamine concentrations of the culture medium. In all experiments, accumulation of dry matter and protein content increased when the sucrose concentration of the culture medium was increased from 1.5 to 150 mM: however, a relatively greater enhancement of dry matter than of protein accumulation resulted in a lower protein concentration at 150 than at 1.5 mM sucrose. Both content and concentration of protein were increased by the increases in glutamine supply to concentrations exceeding 68% protein at 120 mM glutamine. In combination with 150 mM sucrose, however, oil increased as glutamine supply was increased from 0.6 to 6 mM and then decreased as glutamine supply was increased from 6 lo 120 mM. Varying the concentration of sucrose available during seed development also affected embryo composition. Decreased availability of sucrose during either the early or late portion of the culture period resulted in lower accumulation of dry mailer as well as oil. Protein concentration was actually higher for embryos transferred from 150 to 1.5 nM sucrose than for those remaining in 150 mM throughout the culture period: however, the greater percentage of protein was due lo a decrease in accumulation of dry weight. In addition, embryo composition was affected by altering the availability of glutamine during culture, indicating that variation in the level of nitrogen assimilate delivered during seed development can change embryo composition. Decreasing the glutamine concentration of the medium lowered both protein and oil content. In contrast, increasing the glutamine concentration of the medium from 0.6 to 6 mM 8 days after initiation of culture increased the protein content and concentration of the embryo while oil content was not affected.     

Effects of Salt Stress on Carbohydrate Metabolism in Desert Soil Alga Microcoleus vaginatus Gom.

The effects of salt stress on carbohydrate metabolism in Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crusts, were investigated in the present study. Extracellular total carbohydrates and exopolysaccharides （EPS） in the culture medium produced by M. vaginatus increased significantly during the growth phase and reached a maximum during the stationary phase. The production of extracellular carbohydrates also significantly increased under higher salt concentrations, which was attributed to an increase in low molecular weight carbohydrates. In the presence of NaCI, the production of cellular total carbohydrates decreased and photosynthetic activity was impaired, whereas cellular reducing sugars, water-soluble sugars and sucrose content and sucrose phosphate synthase activity increased, reaching a maximum in the presence of 200 mmol/L NaCI. These parameters were restored to original levels when the algae were transferred to a non-saline medium. Sodium and K＋ concentrations of stressed cells decreased significantly and H＋-ATPase activity increased after the addition of exogenous sucrose or EPS. The results suggest that EPS and sucrose are synthesized to maintain the cellular osmOtic equilibrium between the intra-and extracellular environment, thus protecting algal cells from osmotic damage, which was attributed to the selective exclusion of cellular Na＋ and K＋ by H＋-ATPase.     

Optimization of a cultural medium for bacteriocin production by Lactococcus lactis using response surface methodology.

The medium composition for bacteriocin production by Lactococcus lactis ATCC 11454 was optimized using response surface methodology. The selected six factors based on CM medium were sucrose, soybean peptone, yeast extract, KH(2)PO(4), NaCl, and MgSO(4).7H(2)O. Fractional factorial designs (FFD) and the path of steepest ascent were effective in searching for the main factors and approaching the optimum region of the response. By a 2(6-2) FFD, sucrose, soybean peptone, yeast extract, KH(2)PO(4) were found to be significant factors and had positive effects on cell growth, however, only soybean peptone and KH(2)PO(4) were shown to be the two significant factors for bacteriocin production and had negative and positive effects, respectively. The effects of the two main factors on bacteriocin production were further investigated by a central composite design and the optimum composition was found to be 1% sucrose, 0.45% soybean peptone, 1% yeast extract, 2.84% KH(2)PO(4), 0.2% NaCl, and 0.02% MgSO(4) x 7H(2)O. The optimal medium allowed bacteriocin yield to be doubled compared to CM medium.