前体物对石蒜悬浮细胞生长和生物碱积累的影响

为探究苯丙氨酸、酪氨酸和酪胺3种前体物对石蒜悬浮细胞系生长和生物碱积累的影响。通过向培养基添加不同浓度的3种前体物,以及同时添加苯丙氨酸和酪氨酸,考察其对细胞生长量及细胞中生物碱累积的影响。结果表明:苯丙氨酸对细胞的生长和生物碱的积累影响不明显;酪氨酸和酪胺作用显著:添加200μmol/L酪氨酸,细胞中生物碱的含量是对照组的2.56倍,其中力可拉敏和加兰他敏含量为3.77 mg/g和4.46 mg/g,分别是对照组的6.61倍和6.97倍;添加200μmol/L酪胺,细胞中生物碱含量是对照组的2.63倍,力可拉敏和加兰他敏含量为4.45 mg/g和5.14 mg/g分别是对照组的9.08倍和9.18倍;在200μmol/L酪氨酸的基础上添加苯丙氨酸没有明显的增效作用。表明添加酪氨酸和酪胺对细胞生长及生物碱生物合成具有显著的促进作用     

加兰他敏在忽地笑营养器官中的定位(简报)

石蒜属植物为具有地下鳞茎的多年生草本植物．有重要的药用价值,全属植物约有20余种。我国有石蒜属植物约15种,集中分布于长江中下游地区,野生资源比较丰富。忽地笑（Lycorisaurea Herb．）是石蒜属植物在我国分布较广泛的一种。加兰他敏（Galanthamine）等生物碱是石蒜属植物中主要的药用成分。[第一段]     

不同培养基及其元素组成对西洋参愈伤组织悬浮培养物生长和皂苷含量的影响

对MS、67-V和FOX 3种基本基质对西洋参(Panax quinquefolium Linn.)愈伤组织悬浮培养物生长和皂苷含量的影响进行了比较。在3种基本基质中,培养物的鲜重和干重增加量差异不大,而皂苷含量和产量差异较大,其中MS较高,FOX次之,67-V最低。探讨了MS基质中,KNO3、CaCl2和MgO4对培养物生长和皂苷含量的影响。KNO3浓度在237．5mg/L时有利于培养物生长,而浓度在1900mg/L时有利于皂苷合成;CaO2浓度在55．35mg/L时有利于培养物生长,而浓度在332．1mg/L时有利于皂苷合成;MgSO4浓度为92．50mg/L时培养物生长最好,皂苷产量也最高。     

CdCl2对豌豆种子萌发和幼苗生长的影响

以豌豆为实验材料,采用水培方法研究了Cd2 单盐胁迫对豌豆种子萌发与生长的影响。结果显示:(1)Cd2 质量浓度≤1 mg/L时,促进种子萌发,Cd2 质量浓度达到5 mg/L时抑制种子的萌发。(2)随Cd2 质量浓度的增加Cd2 对幼苗根生长的抑制作用逐渐增强;Cd2 质量浓度≤5 mg/L时,促进茎的生长,≥10 mg/L时,抑制茎的生长;且Cd2 对幼苗根生长的抑制作用大于茎。(3)低浓度Cd2 能促进幼苗叶绿素合成,当Cd2 质量浓度高于1 mg/L时,则对幼苗叶绿素合成有抑制作用,且随Cd2 质量浓度增加叶绿素含量逐渐下降。(4)Cd2 诱发的胚根细胞核、染色体畸变率随着Cd2 质量浓度增加而增大。(5)过氧化物酶(POD)同工酶的活性随着Cd2 质量浓度升高而明显增强,Cd2 质量浓度为1 mg/L时POD活性最强,但当Cd2 质量浓度达10 mg/L时,POD的灰度值明显下降。     

黄腐酸对单针藻生长和油脂含量的影响

为了研究不同浓度的黄腐酸对单针藻Monoraphidium sp.FXY-10细胞生长、油脂合成的影响,研究于Kuh1培养基中添加4种不同浓度的黄腐酸(40、80、120和160 mg/L),优化出异养培养条件下最适合藻细胞生长的黄腐酸浓度;并采用黄腐酸与异养-自养两步培养联用的方法提高细胞量和油脂含量,自养培养时在培养基中添加5、25、125和625 mg/L的黄腐酸诱导油脂的合成。结果表明,80 mg/L的黄腐酸对细胞生长的促进作用最显著,细胞量可达6.4 g/L,为对照组的1.5倍。黄腐酸的浓度增加至160 mg/L,藻细胞的生长受到明显的抑制。自养培养阶段,添加25 mg/L的黄腐酸能显著地提高藻细胞的油脂含量,其油脂含量从30.78%增加至54.65%。黄腐酸对于单针藻的生长和油脂合成具有明显的促进作用,黄腐酸与两步法联用在提高微藻细胞量和油脂含量方面具有较好的应用前景。     

用改良CsCl密度梯度离心法提取石蒜叶片和鳞茎总RNA

石蒜[Lycoris radiata（L’Her．）Herb．]是重要的药用植物,其次生代谢产物加兰他敏（galantamine）在临床上的应用非常广泛,是治疗阿尔茨海默氏病的首选药物之一。尽管有关加兰他敏化学合成途径已有许多报道,但由于纯化学合成的成本太高,目前仅限于实验室合成,尚不能用于工业化生产。提高石蒜本身的加兰他敏含量是获得加兰他敏最为经济和环保的方法。因此,研究加兰他敏生物合成的分子机制对于提高其含量具有重要意义。     

Burkholderia sp. IDO3中靛蓝合成基因的克隆表达及其合成特性

【目的】克隆和表达靛蓝合成基因,并将其用于靛蓝合成研究。【方法】对菌株Burkholderia sp.IDO3中靛蓝合成基因进行克隆和大肠杆菌异源表达,构建能合成蓝色色素的基因工程菌。利用液相色谱和质谱对产物进行分析,采用单因素法对培养温度、转速、培养基成分等进行优化,并考察优化条件下的靛蓝合成曲线。【结果】构建了一株重组大肠杆菌E.coli IND_AB,该菌株能够在LB培养基生长的过程中合成蓝色色素,产物分析表明该色素为靛蓝;菌株IND_AB在30°C和150 r/min条件下能在LB培养基中合成22.9 mg/L靛蓝,优化培养条件后产量达到25.4 mg/L;优化LB培养基各组分浓度后产量可提高到35.1 mg/L;外加50.0 mg/L吲哚或0.1 g/L色氨酸后靛蓝产量可分别提高到57.7 mg/L和64.4 mg/L,相比初始产量提高了152.0%和181.2%;靛蓝合成曲线表明在添加吲哚或色氨酸的培养基中,菌株IND_AB前6 h没有靛蓝生成,6-15 h为靛蓝合成加速期,18 h达到产量平衡。【结论】重组大肠杆菌IND_AB可用于生物合成高纯度靛蓝,为靛蓝的微生物合成提供了有效的基因资源。     

镉、铜和锌胁迫下黑藻活性氧的产生及抗氧化酶活性的变化研究

研究了不同Cd、Cu、Zn处理浓度对黑藻体内活性氧()产生及对抗氧化酶(SOD、POD、CAT)活性的分子毒理学效应以探讨高等水生植物抗氧化酶对重金属胁迫的反应。结果表明,三种重金属都不同程度地加快了产生速率;Cu使SOD、POD、CAT活性下降;Cd也都减弱了SOD和POD活性,而CAT活性在0.5—5mg/L处理浓度时增加;Zn对SOD活性也为抑制作用,当浓度为0.5—5mg/L时POD和CAT活性都上升。关联度分析发现Cd、Cu和Zn胁迫下黑藻起主要保护作用的分别为SOD、POD和CAT,而SOD最易受到影响。Cd、Cu处理下的叶绿素含量也都呈下降趋势,而0.5—5mg/L的Zn浓度刺激了叶绿素合成。所有Zn处理、0.5mg/L的Cu处理和0.5—1mg/L的Cd处理的叶绿素a/b值都大于对照值。除了Cu使可溶性蛋白含量减少外,0.5—5mg/L的Zn和0.5—1mg/L的Cd都使其含量增加。综合起来,Cu的毒性最强,其次为Cd,Zn最弱。致死阈浓度分别为:Cu:0.5—1mg/L;Cd:1—2mg/L;Zn:5—6mg/L。SOD是评价重金属对沉水植物毒性效应的灵敏指标。黑藻对水环境Cu污染反应敏感。       

间苯二酚与邻苯二酚对泥鳅血清卵黄蛋白原的诱导作用

为探讨间苯二酚(m-dihydroxybenzene)与邻苯二酚(o-dihydroxybenzene)对雄性泥鳅Misgurnus anguillicau-datus血清卵黄蛋白原(Vitellogenin,Vtg)的诱导作用,将雄性泥鳅分别暴露于4种不同质量浓度间苯二酚(5.287mg/L、6.531mg/L、8.072mg/L、10.000mg/L)和邻苯二酚(16.708mg/L、17.742mg/L、18.836mg/L、20.00mg/L)中持续7d、14d、21d和28d,采用碱不稳定性蛋白结合磷法检测血清Vtg水平的变化。结果显示,与对照组相比,泥鳅在低质量浓度间苯二酚(5.287mg/L)和邻苯二酚(16.708mg/L)中暴露7d时,血清Vtg水平均有极显著升高,并且随着暴露浓度的增加,Vtg水平逐渐升高;在同一暴露浓度下,Vtg水平随暴露时间的延长逐渐升高;2种酚类化合物相比,暴露在间苯二酚中的泥鳅血清Vtg水平高于邻苯二酚。上述结果说明,2种化合物均可诱导雄性泥鳅肝脏合成Vtg,具有一定的环境雌激素效应,其诱导作用随着暴露剂量的增大和时间的延长逐渐增强。     

4种常用水产药物对青虾幼虾的毒性研究

采用静水法研究了4种常用水产药物对青虾幼虾的毒性。结果表明：水温29℃~30℃时,溴氯海因对青虾幼虾24 h、48 h和72 h的LC50分别为19.05 mg/L、11.75 mg/L、11.75 mg/L,安全浓度为1.24 mg/L;聚维酮碘对青虾幼虾的24 h、48 h和72 h的LC50分别为49.27 mg/L、44.61 mg/L、43.68 mg/L,安全浓度为10.52 mg/L;甲醛对青虾幼虾的24 h、48 h和72 h的LC50分别为72.47 mg/L、66.41 mg/L、53.71 mg/L,安全浓度为10.93 mg/L;高锰酸钾对青虾幼虾的24 h、48 h和72 h的半致死浓度（LC50）分别为6.06 mg/L、4.07 mg/L、3.79 mg/L,安全浓度为0.48 mg/L。4种药物对青虾幼虾的毒性依次为高锰酸钾〉溴氯海因〉聚维酮碘〉甲醛。     

Adventitious shoot regeneration from in vitro leaf explants of <Emphasis Type="Italic">Fraxinus nigra</Emphasis>

In order to establish an attractive method for the production of valuable medicinal alkaloids (galanthamine and lycorine), the plants of Leucojum aestivum and L. aestivum ‘Gravety Giant’ grown in bioreactor RITA® were subjected to various concentrations of methyl jasmonate (MeJA), salicylic acid (SA), 1-aminocyclopropane-1-carboxylic acid (ACC) and 2-chloroethylphosphonic acid (ethephon) at different times of culture. The application of MeJA showed a negative effect on L. aestivum and L. aestivum ‘Gravety Giant’ plant growth. We observed that the incubation of plants during 168 h with 100 µM of MeJA resulted above two times lower F.W. (fresh weight) increments compared with control. While SA showed an inhibitory effect only on the growth of L. aestivum cultures. ACC and ethephon had a positive effect on both types of culture. Treatment with 50 µM of MeJA during 168 h stimulated galanthamine and lycorine biosynthesis in L. aestivum and L. aestivum ‘Gravety Giant’ cultures. In addition, the accumulation of galanthamine was increased when 10 µM of ACC were added to both types of culture. 10 µM of ACC stimulated also lycorine biosynthesis by L. aestivum ‘Gravety Giant’. The addition of 10 µM of ethephon had a positive effect only on lycorine production in plants of L. aestivum. SA promoted galanthamine and lycorine biosynthesis in tested plants. Indeed the highest galanthamine (0.8 mg/g dry weight: D.W.) and lycorine (1.53 mg/g D.W.) concentrations were observed in L. aestivum ‘Gravety Giant’ plants treated with 5 µM of SA during 10 h.     

Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC₅₀ = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.     

Elicitation of galanthamine biosynthesis by Leucojum aestivum liquid shoot cultures

The effects of methyl jasmonate and jasmonic acid on galanthamine production, phenolic acid content and growth of Leucojum aestivum L. shoot culture, cultivated in submerged conditions were investigated. The best time-point for addition of elicitors was during the exponential phase of the culture growth. The maximal contents of galanthamine and lycorine (226.9 μg/flask and 491.4 μg/flask, 1.36 and 1.67-fold higher compared to the control, respectively) were achieved after elicitation with jasmonic acid, whereas the elicitation with methyl jasmonte resulted in maximal accumulation of phenolic acids. It was demonstrated that the boosting effect of jasmonic acid on Amaryllidacea alkaloid biosynthesis was due to induction of the activity of tyrosine decarboxylase, whereas methyl jasmonate stimulates the biosynthesis of phenolic acids by inducing mainly the activity of phenylalanine ammonia-lyase.     

The geographic isolation of Leucojum aestivum populations leads to divergation of alkaloid biosynthesis

Leucojum aestivum, an industrial source of the acetylcholinesterase inhibitor galanthamine, shows a great chemodiversity in its alkaloid synthesis. Samples from various geographically distinct populations from Bulgaria and the Balearic Islands were studied by GC–MS. The alkaloid pattern of the plants of L. aestivum (subsp. pulchellum) from the Balearic Islands were dominated by crinine type compounds. Populations of homolycorine chemotype were distributed along the Danube river in the north part of Bulgaria, which is separated from the south part by the Balkan mountains. Populations with high accumulation of lycorine were found in East Bulgaria near the sea coast, while the south populations were dominated by galanthamine type synthesis. The average of the galanthamine content was found to vary from 0.003 to 0.08% (referred to dry weight) in the north, and up to 0.42% in the southern Bulgarian populations. Some individuals showed up to 0.65% galanthamine. The galanthamine content of the plants from the Balearic island was 0.1% of DW. The galanthamine percentage in the total alkaloid mixture ranged from 0.2 to 95% of the total alkaloids. Our study demonstrated that the geographic isolation of the populations of L. aestivum has led to divergation in the alkaloid biosynthesis and consequently to the occurrence of different chemotypes. This chemodiversity in both alkaloid patterns and galanthamine content provides an opportunity for further selection work toward a galanthamine-rich crop, on the one hand, and makes the species an excellent biological system for molecular studies leading to further improvement of the galanthamine production, which is a valuable alkaloid used in medicine for the treatment of Alzheimer's disease.     

Stimulating effect of both 4’‐O‐methylnorbelladine feeding and temporary immersion conditions on galanthamine and lycorine production by Leucojum aestivum L. bulblets

Bulb cultures of Leucojum aestivum and L. aestivum ‘Gravety Giant’ were subcultured in medium containing the precursor 4’‐O‐methylnorbelladine (MN) at various concentrations [0 (control), 0.15 and 0.3 g/L]. The cultures were conducted in bioreactor RITA® and lasted for 15, 30, 40 and 50 days. The growth rate and the alkaloid accumulation in bulblets were studied. For this latter purpose, a purification method was developed. It comprised a highly selective solid phase extraction using on the one hand, UPTI‐CLEAN SI and SCX cartridges for plant extracts and on the other hand, 2H cartridges for culture media. Pure alkaloidal fractions were, thus, analyzed by LC‐ESI‐MS allowing the quantitative evaluation of galanthamine and lycorine from culture extracts. Precursor feeding along with temporary immersion conditions was found to significantly improve the accumulation of both galanthamine and lycorine. The maximal concentrations of galanthamine (0.81 mg/g DW) and lycorine (0.54 mg/g DW) in L. aestivum bulblets were reached, respectively, after 40 days of culture with 0.15 g/L of precursor and after 30 days of culture with 0.3 g/L of precursor. In L. aestivum ‘Gravety Giant’ bulb cultures, 0.3 g/L of precursor was the best condition for both galanthamine (0.6 mg/g DW after 50 days) and lycorine (1.13 mg/g DW after 30 days).     

Galanthamine production in “shoot-clump” cultures of Narcissus confusus in liquid-shake medium

Galanthamine (GAL) is increasingly used in the treatment of Alzheimer's disease. We have attempted to develop a method of producing this alkaloid using in vitro cultures of Narcissus confusus plants. The “shoot-clump” culture in liquid medium was shown to be an appropriate method for the micropropagation of this bulbous plant. The complete process included three steps:

1. culture of “twin-scales” starting from the bulbs;
2. culture of the newly formed shoots in a medium for bud proliferation (Murashige Skoog+1 mg l^-1 of 2,4-dichlorophenoxyacetic acid+5 mg l^-1 of benzyladenine), and
3. culture of “shoot-clumps” in a liquid-shake medium. Here we describe the effect of the addition of trans-cinnamic acid, a precursor in the biosynthesis of the Amaryllidaceae alkaloids, on the production of galanthamine and related alkaloids, and also on the growth of the “shoot-clump” culture. The production of galanthamine was found to be inhibited by the addition of the precursor, which promoted the production of the other alkaloid in the same biosynthetic pathway, N-formyl-norgalanthamine. The total production of galanthamine in the control cultures in day-long photoperiod was 2.50 mg per culture, of which 1.97 mg per culture were released into the medium.

     

In sito galanthamine extraction during the cultivation of Leucojum aestivum L. shoot culture in two‐phase bubble column cultivation system

Two‐phase bioreactor cultivation system was developed and applied for in sito recovery of extracellular galanthamine during the cultivation of Leucojum aestivum L. shoot culture in a modified column bioreactor system. The inclusion of an external circulation column with adsorbent resin Amberlite XAD‐4 as a second phase, on the 21st day of the beginning of cultivation resulted in 1.25 folds increase in biomass accumulation and maximal amounts of accumulated galanthamine of 6 mg/L (3.1 mg/L intracellular and 2.9 mg/L extracellular). It was demonstrated that the inclusion of a second phase at the cultivation of the L. aestivum shoot culture in a bubble column bioreactor with internal sections redirected the alkaloid metabolism to galanthamine synthesis and inhibits the synthesis of hemanthamine and lycorine type alkaloids. Our research demonstrated that the application of the two‐phase cultivation systems could be an important tool to increase the yields of valuable secondary metabolites in plant tissue culture‐based bioprocess.     

Galanthamine production by Leucojum aestivum in vitro systems

The callus induction from young fruits of Leucojum aestivum was performed on Murashige–Skoog nutrient medium supplemented with 4 mg/L 2,4 dichlorphenoxyacetic and 2 mg/L 6-benzylamynopurine. Further, by planting the obtained calluses on the same nutrient medium supplemented with 1.15 mg/L α-naphtylacetic acid and 2.0 mg/L 6-benzylamynopurine shoot cultures were established. The growth and galanthamine accumulation of obtained L. aestivum in vitro systems were studied. It was established that the amount of accumulated galanthamine strongly depended on the level of the differentiation. The maximum yield of biomass (17.8 g/L) and the maximum amount of accumulated galanthamine (2.5 mg/L) were achieved after day 35 of submerged cultivation of L. aestivum 80 shoot culture, performed under illumination. Data concerning the time courses of the utilization of the main nutrient components of the medium during cultivation of L. aestivum shoot culture are presented as well.     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

A novel biotransformation process of 4′-demethylepipodophyllotoxin to 4′-demethylepipodophyllic acid by Bacillus fusiformis CICC 20463, Part II: process optimization

This work optimized the novel biotransformation process of 4′-demethylepipodophyllotoxin (DMEP) into 4′-demethylepipodophyllic acid (DMEPA) by Bacillus fusiformis CICC 20463. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/L of yeast extract and 10 g/L of peptone were optimal for DMEPA production (i.e., 2.81 ± 0.21 mg/L), while not beneficial for the cell growth of B. fusiformis. This indicated that the biosynthesis of DMEPA was not corresponded well to the cell growth of B. fusiformis. 40 g/L of sucrose was optimal for DMEPA production (i.e., 2.94 ± 0.17 mg/L), and 3 g/L of NaCl was the best for DMEPA production (i.e., 4.10 ± 0.18 mg/L). Secondly, the production of DMEPA was significantly enhanced by the control of substrate concentration and culture pH. 100 mg/L of substrate was optimal for DMEPA production (i.e., 6.47 ± 0.35 mg/L), and DMEPA concentration was enhanced to 38.78 mg/L by controlling culture pH at 9.0 in the stirred-tank bioreactors. The fundamental information obtained in this study provides a simple and efficient way to produce DMEPA by biotransformation.