脂氧合酶在诱导红豆杉细胞产紫杉醇中的作用

对红豆杉悬浮培养细胞中脂氧合酶(LOX)在诱导子诱导紫杉醇合成中的作用进行了探讨。结果表明真菌诱导子处理可提高细胞内LOX的活性和紫杉醇的产量,而诱导前用LOX抑制剂菲尼酮处理,可完全抑制诱导子对LOX活性和紫杉醇合成的诱导作用。说明LOX途径可能参与了紫杉醇的合成过程。外加茉莉酸甲酯也可激活LOX活性和紫杉醇合成,诱导前用菲尼酮处理可抑制诱导子诱导的LOX活性和紫杉醇合成,说明外源茉莉酸甲酯可能是通过激活细胞内LOX途径而启动下游紫杉醇的合成。为了进一步研究脂氧合酶在紫杉醇合成中的作用。我们还对红豆杉细胞脂氧合酶的分布和分子量等性质进行了研究。     

基于定量PCR技术探讨紫杉醇生物合成的限速步骤

次生代谢产物牛物合成受到发育和诱导的调控,本实验研究了组织分化和诱导处理对紫杉醇生物合成的影响,并采用定量PCR技术分析了紫杉醇生物合成不同阶段关键酶基因的动态表达特征。结果表明。紫杉醇主要分布在中国红豆杉（Taxus chinensis）树皮和根皮组织中,针叶内含量很少,催化紫杉醇功能官能团连接的关键酶摹因也主要定位在树皮和根皮组织巾;茉莉酸甲酯（MJ）和真菌诱导子F5分别提高了中国红豆杉悬浮培养细胞HG-1紫杉醇得率8倍和10倍,同时有效诱导紫杉醇生物合成基因的表达。发现催化紫杉醇侧链连接的基因与紫杉醇生物合成早正相关。结果表明。紫杉醇生物合成的限速步骤是催化功能官能团连接的步骤。     

水杨酸对真菌诱导子诱导红豆杉悬浮细胞膜脂过氧化和紫杉醇生物合成的影响

研究了 5 0 mg· L- 1真菌诱导子 (F5) ,5 0 mg· L- 1水杨酸 (SA) ,5 0 mg· L- 1F5+5 0 mg· L- 1SA3种处理 ,对红豆杉悬浮细胞膜脂过氧化和紫杉醇合成的影响。结果表明 :F5和 SA单独处理红豆杉细胞均引起细胞膜脂过氧化。SA+F5联合处理可以减轻 F5单独处理细胞所引起的膜脂过氧化程度 ,SA+F5联合处理与真菌诱导子处理相比 ,较大地提高了过氧化物酶的活性 ,得到较多的生物量。3种处理方法均可提高红豆杉细胞紫杉醇产量 ,特别以 F5+SA处理得到产量最高 ,达到 1 1 .5 mg· L- 1,分别为 F5,SA和对照组的 1 .5倍、2 .0倍和7.5倍。结果显示 :在真菌诱导子诱导与水杨酸的联合作用下提高紫杉醇产量 ,可能与水杨酸减轻真菌诱导子所引起的细胞膜脂过氧化程度有关     

内生真菌和诱导子对长春花悬浮细胞及生物碱合成的影响

目的:探讨内生真茵和诱导子对长春花悬浮细胞生长及生物碱合成的影响.方法:接种内生真茵与悬浮细胞的共同培养,添加诱导子到细胞培养液中对长春花悬浮细胞进行诱导处理,检测实验处理后长春花悬浮细胞各项生化指标.结果:培养液pH值上升,悬浮细胞MDA舍量增加,细胞抗氧化酶(POD、CAT)和生物碱合成的关键性酶(PAL、TDC)活性升高.生物碱产量得到提高,诱导组悬浮细胞生物碱产量达到770.36μg/gFW,共培养组生物碱产量为693.76 μg/gFW,分别比对照组提高了48%和32%.结论:真菌及其诱导子能改变长春花悬浮培养细胞的态势,导致细胞代谢结构的改变,使生物碱的产量提高.     

强恒磁场对红豆杉细胞分裂和紫杉醇含量的影响

研究了磁场对红豆杉细胞分裂和紫杉醇含量的影响。结果表明,0.4T磁场处理3,4d对红豆杉细胞活力无显著性影响;各实验组中,预培养3d,磁场处理3d,细胞分裂指数提高了162％;加入诱导子后,紫杉醇含量较对照提高115％。     

植物细胞工程技术生产紫杉醇研究进展

紫杉醇是一种二萜类生物碱,主要从红豆杉树皮中分离提取。目前采用植物细胞工程技术是提高紫杉醇产率、保护稀缺资源红豆杉、解决紫杉醇药源紧缺的一种最有效方法。该文对近十年来国内外有关采用植物细胞工程技术生产紫杉醇的研究进展,包括红豆杉细胞系的建立、悬浮培养条件的研究、生物反应器培养以及紫杉醇合成代谢调控方面的最新研究进展进行综述。并重点论述了前体、诱导子和代谢支路抑制剂对红豆杉细胞悬浮培养生产紫杉醇的影响,为植物细胞工程技术生产紫杉醇提供借鉴和参考。     

美丽镰刀菌培养液对东北红豆杉悬浮细胞防御反应及紫杉醇合成的影响

美丽镰刀(Fusarium mairei)是一分离自南方红豆杉(Taxus chinensis var.mairei)的产紫杉醇内生真菌,用B5培养基培养6 d,去菌尘幺后制得美丽镰刀菌培养液,并从中提取其胞外多糖.研究了4%(V/V)美丽镰刀菌培养液及4%(W/V)胞外多糖两种处理,对东北红豆杉(T.cuspidata)悬浮细胞防御反应及紫杉醇合成的影响.结果表明:两种处理均能诱导东北红豆杉细胞的防御反应.但美丽镰刀菌培养液的影响明显大于胞外多特(P<0.05).另外,两种处理均可促进东北红豆杉细胞紫杉醇的合成与释放.美丽镰刀菌培养液处理得到的紫杉醇与其释放率分别是对照的2.5倍8.8倍,而胞外多糖处理得到的紫杉醇与其释放率则分别是对照的1.5倍与3倍.     

红豆杉细胞与赤霉菌交互诱导对红豆杉细胞紫杉醇含量的影响

在赤霉菌培养过程中加入红豆杉细胞诱导物,再用此赤霉菌诱导悬浮培养的红豆杉细胞。与未受植物来源物质作用的单向诱导相比,该交互诱导使红豆杉细胞的紫杉醇含量提高5倍,交互诱导的效果受诱导物的各类的影响,其中,诱导物是红豆杉细胞壁和赤霉菌细胞壁时,交互诱导效果最好。     

红豆杉细胞培养的研究

从红豆杉（Ｔａｘｕｓｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇ）Ｒｅｈｄ）的嫩茎及针叶诱导的出愈伤组织,对愈伤组织培养及细胞悬浮培养进行了研究,利用ＨＰＬＣ方法测定它们合成紫杉醇的能力,发现了能够提高培养细胞生长速率及紫杉醇含量的一些因子,红豆杉愈伤组织及悬浮培养细胞的生长速率已分别达到０．２５ｇ／Ｌ．ｄ和０．２８ｇ／Ｌ．ｄ。而他们的紫杉醇含量分别是０．００２６％和０．０１２％。     

寡聚糖诱导悬浮培养南方红豆杉细胞的凋亡(英)

在真菌 (Fusariumoxysporumf.vasinfectum (Atkinson)SnyderetHansen)寡聚糖诱导悬浮培养南方红豆杉(Taxuschinensis (Pilger)Rehd .var.mairei (LemeeetL啨ｖｌ.)ChengetL .K .Fu)细胞生产紫杉醇的体系中发现细胞出现凋亡 ,次生代谢增强。电镜观察到细胞核质和原生质出现凝集现象 ,液泡内出现大量的高电子致密体。核DNA经琼脂糖凝胶电泳 ,呈 2 0 0bp的整数倍的梯状条带 (ladders) ;而对照组细胞核DNA完整 ,呈大片段 ,细胞完整 ,细胞器发达 ,但紫杉醇合成速率很低。加入寡聚糖后 ,细胞防御系统开启 ,细胞生长停止 ,次生代谢物酚类物质大量积累且次生壁加厚 ,多酚氧化酶活性迅速提高 ,苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在 1h后急速提高 ,目的产物紫杉醇在诱导后 72h达到峰值 ,比对照组提高了 6倍 ,且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性。     

Hydrogen peroxide from the oxidative burst is not involved in the induction of taxol biosynthesis in Taxus chinensis cells

In cell suspension cultures of Taxus chinensis, 40 mg/l fungal elicitor from Aspergillus niger and 20 microM HgCl2 elicited 5.7 and 3.6 mg/l taxol, which was a 9-fold and 5-fold increase vs. compared with the control, respectively. The fungal elicitor induced hydrogen peroxide (H2O2) accumulation but HgCl2 did not, indicating that H2O2 was not necessary for enhancement of taxol induced by elicitor. Compared with the treatment with fungal elicitor alone, exogenous catalase, ascorbic acid, diphenylene iodonium and superoxide dismutase induced a 0.45, 0.4, 0.7 and 1.4-fold H2O2, but elicited taxol production, which was 0.98, 1.2, 1.1 and 0.9-fold, respectively, vs. non-treated cells Elicitor-induced taxol production was not accorded with the amount of H2O2 production.     

Oxidative Stress and Taxol Production Induced by Fungal Elicitor in Cell Suspension Cultures of Taxus Chinensis

Treatment of Taxus chinensis cell suspension cultures with fungal elicitor resulted in an oxidative stress characterized by H₂O₂ production, malondiadehyde (MDA) accumulation and cell death. This oxidative stress was dependent on the concentration of elicitor. Cells exposed to elicitor accumulated taxol, however, not proportional to elicitor concentration. High production of taxol occurred in cells treated with the suitable elicitor concentration. We concluded that oxidative stress had the deleterious effect on taxol production. Simultaneous treatment with elicitor and ascorbic acid (ASA) changed the oxidative stress and taxol production. Production of taxol in cells treated with 200 mg dm^–3 elicitor and ASA was enhanced compared with that in cells treated with only 200 mg dm^–3 elicitor, while production of taxol in cells treated with 100 and 50 mg dm^–3 elicitor and ASA was decreased compared with that in cells treated with 100 and 50 mg dm^–3 elicitor.     

茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞中紫杉醇含量的影响

比较了茉莉酸甲酯与真菌诱导物、水杨酸组合对红豆杉细胞几个抗病相关指标（POD、CAT活力、H2O2含量）及紫杉醇含量的影响,3种信号分子的组合对POD、CAT、H2O2及紫杉醇含量的影响是不一致的,MJ单独添加,MJ与SA联合作用以及MJ与F5联合作用都可使POD活力增加,且12h后H2O2含量均升高,约在48h达到高峰,为对照的2倍左右,但72h后,MJ单独添加和MJ与SA联合作用组中H2O2含量变化不大,F5与MJ联合作用则使H2O2含量持续比对照高。MJ单独添加使CAT酶活在144h后才较对照低,F5、SA的加入都可使CAT酶活下降,SA的作用更显著。说明三者的诱导途径并不完全一样,以SA和MJ联合添加对紫杉醇合成的促进作用最大,含量达到细胞干重的0．04％。     

真菌诱导子对茶条槭细胞没食子酸积累的影响

茶条槭（Acer ginnala Maxim.）叶片中含有没食子酸,但含量较低。真菌诱导子可以增加植物中一些次生代谢产物的含量,但其机理尚不十分清楚。本研究在茶条槭细胞悬浮培养的对数期加入内生真菌（Phomopsis sp.）诱导子,茶条槭细胞中没食子酸含量在24 h后开始增加,48 h时没食子酸含量达到峰值,最高含量为12.2 mg·g^-1 DW,是对照的1.58倍。茶条槭细胞对内生真菌诱导子的防御反应不同于对病原和非生物胁迫。真菌诱导子不提高培养液中pH值,也不明显增加胞内Ca²⁺浓度,但增大细胞膜通透性。培养液电导率差异显著,细胞核发生分裂,说明真菌诱导子可能促进茶条槭细胞核内有丝分裂,促使茶条槭细胞对培养液中的无机盐离子的吸收,以满足细胞生长的需要。PAL酶活性升高,在48 h时为对照的1.75倍,说明PAL酶可能参与了真菌诱导没食子酸的合成。     

Vanadate mimics effects of fungal cell wall in eliciting gene activation in plant cell cultures

Cell-suspension cultures of peanut (Arachis hypogaea L.) can be used as a very sensitive and rapidly responding physiological system for monitoring extracellular signals. Elicitors effect the activation of the genes that code for a set of enzymes synthesizing stilbenes. Within 2–6 h after administering micromolar, concentrations of orthovanadate to the suspended cells, the enzyme activities of phenylalanine ammonia-lyase, stilbene synthase, and cinnamate 4-hydroxylase increased 10-to 100-fold. The transient time course of induction, and the quality and quantity of gene expression found with vanadate as artificial elicitor were very similar to those observed after biotic stress generated by fungal cell walls. The dose-response of vanadate as an elicitor of gene expression in intact cells matched precisely its inhibitory effect on the ATPase activity of isolated plasma membrane. By concentrating, on the profiles of cinnamate 4-hydroxylase activity, we observed differences between the effects elicited by fungal cell wall or vanadate when different stages of cell development were analyzed. Unlike the fungal elicitor, vanadate did not induce the hydroxylase activity when cells at the stationary phase of the cell cycle were used. This lack of response was not the result of a decrease in membrane biosynthesis. The finding, that the effects of vanadate and fungal elicitor are additive indicates that vanadate does not interfere negatively with the perception of the biotic signal but rather addresses the same intracellular intermediate of the signalling process. We hypothesize that membrane potentials created or modulated by ATPases may be intermediates in the signal chain, starting with the recognition process at the plasma membrane and eventually leading to the production of stilbenes as low-molecular-weight plant-defence products.Abbreviations ER endoplasmic reticulum - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol deceased     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in <Emphasis Type="Italic">Taxus chinensis</Emphasis> cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicítor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.     

Enhancement of taxol production and release in Taxus chinensis cell cultures by ultrasound,methyl jasmonate and in situ solvent extraction

This study evaluates the use of a novel mechanical stimulus, ultrasound (US), and a putative chemical elicitor, methyl jasmonate (MJ), combined with in situ solvent extraction (two-phase culture), to enhance taxol production by Taxus chinensis cells in suspension culture. The volumetric taxol yield was increased 1.5- to 1.8-fold with 2 min US treatment once or twice during a 4-week culture period, about 5-fold with 60-120 microM MJ, and 7- to 9-fold by in situ solvent extraction of taxol with dibutyl phthalate (DBP) (11% v/v). The percent of extracellular taxol or taxol release was also significantly increased. The combined use of US (day 5 or 9) and MJ treatment (day 7) resulted in taxol yields 20-50% higher than each of the treatments used alone. The most favorable strategy for taxol production was the application of US or MJ treatment, followed by in situ solvent extraction, giving rise to a taxol yield of 33-35 mg/l, about 17-fold higher than the control, at 1.9 mg/l. It was found that the organic solvent DBP, as well as US and MJ, stimulated the enzyme activity of secondary metabolic pathways, which was partially responsible for the enhanced taxol production.     

Cell-lytic activity of tobacco BY-2 induced by a fungal elicitor from alternaria alternata attributed to the expression of a class I beta-1,3-glucanase gene

Stress-induced cell-lytic activity was found in tobacco BY-2 cells treated with various stresses. Among 14 stresses, an elicitor fraction isolated from Alternaria alternata showed the highest inducing activity. Cell-lytic activity increased for 72 h even in the control sample, treated with distilled water, and several isozymes of beta-1,3-glucanases and chitinases were found to be involved in it. In contrast, cell-lytic activity in BY-2 cells treated with a fungal elicitor reached a higher level after 60 h. The principal enzymes specifically involved in this stress-induced portion are speculated to be basic beta-1,3-glucanases. A class I beta-1,3-glucanase gene (glu1) was found to be the specific gene for the stress-induced cell-lytic activity. Its expression became observable at 24 h, and the intensity reached a maximum at about 60-72 h. The glu1 was thus assigned as a late gene. Its role in the stress response is discussed in conjunction with earlier genes such as chitinases.