发菜(Nostoc flagelliforme)培养条件的研究

研究了光照强度、日供水次数、CO2浓度和培养基成分对发菜生长的影响。结果显示,中度光强(114μmol.m-2.s-1)下发菜生长最快;发菜的生长基本同供水次数成正相关系;CO2浓度的升高并没有显著促进发菜生长,低光条件下(57μmol.m-2.s-1),高浓度的CO2(2800μL/L)抑制了发菜的生长;用BG11培养的发菜生物量的增长显著高于用BG110培养的;BG11培养基中K+和CO32-的缺失并没有显著影响发菜的生长。     

液体悬浮培养条件下发菜细胞的光合速率与呼吸速率

用液相氧电极测定离体悬浮生长发菜细胞的光合速率和呼吸速率的结果表明,发菜细胞的光补偿点为15￣16μmol·m-2·s-1,光饱和点为90μmol·m-2·s-1,光抑制点为190μmol·m-2·s-1。25℃下发菜细胞光合速率最高,呼吸速率则在10￣50℃范围内随温度升高而增强。发菜细胞光合作用的最适pH值为7.0￣7.5,呼吸作用最适pH值为9.0。BG110无氮培养基中添加30mmol·L-1NaNO3,发菜细胞的光合速率增加约20%。培养基中Na2HPO4浓度为1.75mmol·L-1时,细胞光合速率和呼吸速率最大,随后保持稳定。钾盐浓度变化对发菜细胞光合速率和呼吸速率的影响不显著。     

培养液及其浓度和pH值对草履虫种群增长的影响

采用稻草、小麦、奶粉、酵母、玉米粒、荷叶等6种培养液培养草履虫,结果表明,稻草、玉米粒和奶粉培养液为草履虫的理想培养液。另外,对上述3种理想培养液的不同浓度和稻草培养液的不同pH值影响草履虫的生长繁殖进行了实验研究,统计分析表明影响较为明显。稻草培养液的最适浓度为1%~2%;奶粉培养液的最适浓度为0.1%~0.2%;玉米粒培养液的最适浓度为1%~2%;稻草培养液最适pH值为7.0。     

温度,含水量和光照对发菜固氮酶活性的影响

陆生蓝藻发菜(Nostoc flagelliforme)固氮酶活性的最适温度为21—28℃;最适藻丝体含水量为1000—1500%;最大饱和光强达150—200焦耳/m~2·秒。在湿润状态下,发菜对较高的温度很敏感。在45℃下1小时,发菜固氮酶失掉活性。在干燥状态下,发菜在55℃高温下,每天5小时,放置21天后,其固氮酶活性保持不变。预先培养4—5天的湿发菜对干燥变得敏感;但在干湿交替循环中,其固氮酶活性逐步提高,并明显地改善了它对干燥的抵抗能力。此外,在高盐浓度下(0.17—0.43mol/LNaCl)发菜的固氮酶活性被强烈地抑制,说明它不能耐盐碱。发菜的固氮生理特性是它对荒漠草原生态条件适应的结果。干湿交替循环或许是发菜维持生存的必需条件。     

四种外生菌根真菌产生植物激素的研究

在纯培养条件下,利用HPLC法,分别测定了四种外生菌根培养液和菌丝体中的五种植物激素含量。检测结果,除没有脱落酸（ABA）外,玉米素（Z）、玉米素核苷（ZR）、赤霉素（GA3）、吲哚乙酸（IAA）,均有一定含量。培养液中激素含量远高于菌丝体中的含量,GA3在各种激素中含量最高。从培养液中的激素总量看,Suillus属的两个种Suillusgrevillei和Suillusbovinus高于另外两个种Rhizopngonluteolus和Piso     

培养条件下发菜的形态建成

固体培养基培养的发菜(Nostoc flagelliforme)在黑暗与低光强(2·s)条件下细胞发育受到抑制,在光强10、20、30、60 μmol/m2·s 条件下细胞生长良好, 但在60 μmol/m2·s 条件下藻丝体易变黄; 液体充气培养的发菜在光强20、60、180 μmol/m2·s 条件下生长速率、类胡萝卜素与多糖含量均随光强升高而增加。发菜在低营养水平时形成的异形胞较多, 异形胞的发生位置也多样, 有端生、间生和连生。当琼脂浓度为0.5%—4%时发菜具有相同的形态发育特征, 从藻殖段发育至丝状聚集体状态, 再从聚集体中释放藻丝; 当琼脂浓度为6%—8%时发菜发育至丝状聚集体状态, 藻丝包裹在厚胶鞘中, 观察不到藻丝和藻殖段的释放。以上结果表明光照和培养基的含水量对发菜细胞发育具有重要影响。       

干旱胁迫对发菜超微结构及抗性生理的影响

发菜是一种陆生固氮蓝藻,具有强烈的旱生生态适应性。对干旱胁迫条件下发菜超微结构和抗性生理进行了研究。结果表明：随着干旱胁迫加重,发菜细胞大小和细胞壁厚度变化不显著,胶质鞘趋于紧密,类囊体排列趋于紊乱,多角体变得模糊不清甚至消失,糖原颗粒数目减少,但结构颗粒数目没有明显变化。随着干旱胁迫的加重,发菜SOD、CAT活性呈先升高再下降趋势,SOD在含水量为120%时达到高峰;CAT活性在含水量为445%时达到高峰;MDA,氧自由基随着干旱胁迫加重,其含量呈上升趋势;H₂O₂含量随干旱胁迫加重呈先升高再下降趋势,在含水量为120%时达到峰值。干燥储存1年的发菜与恢复活性的发菜有明显差异性,其SOD、CAT活性较低、MDA、H₂O₂含量较少,但是氧自由基含量最高。研究结果对深入研究发菜的耐旱机理奠定了基础。     

发菜的生态条件及其规律分析

 本文研究了甘肃永登和内蒙阿拉善左旗的发菜生态条件,发现了发菜生长中的水分节律现象。 发菜生于干燥的高原荒漠草原地带,属大陆性气候。其年平均气温为4.5—8℃,≥10℃积温为2231—2800℃·年,年温差大,昼夜温差明显。年平均降水量为201—290mm,集中在6—8月。年平均相对湿度为46—58%,有露水的天气约80天。土壤主要是由第三纪红土母质发育而来的棕钙土,营养贫乏,石灰质沉积明显,呈强碱性反应。年日照时数3000±300小时,辐射强烈。发菜在结构和生理上表现出强烈的旱生生态适应性,包括它的耐旱,耐贫瘠,嗜阳、嗜碱以及对温度变化的适应性强。 发菜生长中具有明显的水分节律现象。在5—10月,在暂短的降雨和露水后,藻体迅速吸取水分而得以生长。尔后,水分又很快被蒸发,藻体变干,生长停滞。发菜在这种湿润⇌干燥的反复节律中得到不断的断续生长和积累,从而完成发菜在干燥条件下的年生长过程。     

大气CO_2浓度升高对不同氮生长条件下的两种大型海藻光合作用的影响

以龙须菜和蛎菜两种大型海藻为试验材料,在室外自然条件下培养,设置常规CO2浓度(390μL·L-1)和高CO2浓度(800μL·L-1)空气两种通气条件、以及10和150μmol·L-1两种氮营养盐供应水平,以探讨大气CO2浓度升高对不同氮营养盐生长条件下海藻生长与光合特性的影响。结果表明:不管是高氮还是低氮的培养条件,大气CO2浓度升高都能促进这两种海藻的生长;在龙须菜中,这种生长促进作用在低氮培养条件下更高,而在蛎菜中这种生长促进作用在高氮培养条件下较高;短期海水中高CO2浓度能促进蛎菜和龙须菜的光合作用;长期高CO2浓度生长环境依然能维持龙须菜高的光合作用,但抑制蛎菜的光合作用。另外,不管培养液中CO2供应条件如何,氮加富均促进了龙须菜和蛎菜这两种海藻的光全和呼吸作用。     

妊娠早期人胎盘绒毛组织中hcG分泌功能的调节

本工作研究了在体外无血清培养条件下,不同类型的激素对妊娠6—8周人绒毛组织hCG分泌功能的影响。绒毛组织被剪成约1mm~3的小块,接种在涂有鼠尾胶原的培养瓶内,每瓶加入2ml McCoy’s 5a培养液,于37℃、5％ CO_2条件下预孵48小时以排除内源激素。更换培养液后加入不同浓     

Cell differentiation and colony alteration of an edible terrestrial cyanobacteriumNostoc flagelliforme,in liquid suspension cultures

Morphological characteristics of an edible terrestrial cyanobacterium Nostoc flagelliforme in liquid suspension cultures under photoautotrophic conditions are presented. Different cell forms alternated in a regular manner during the experimentation period (30 d). N. flagelliforme exhibited a very complex life cycle in terms of colony morphology, including mainly 4 different colony morphological forms, viz. hormogonia, filaments, seriate colonies and aseriate colonies. Under laboratory conditions it formed spherical colonies on solid media but not threadlike colonies as it did under natural conditions. The overall life span of the alga was not altered by the existence of different nitrogen sources in the media despite the depression of some cell forms or colony morphologies. Compared with growth on the medium with urea and ammonium as nitrogen sources, the alga on standard medium had a short period of hormogonia and aseriate colony, suggesting that both ammonium and urea could stimulate the formation of hormogonia, at the same time inhibiting the formation of heterocystous cells. The new information on the growth and morphology of N. flagelliforme could be potentially used for the scale-up or field cultivation.     

水分对发状念珠藻生理活性的影响

研究了水分对发状念珠藻(Nostor flagelliforme Born.et Flah.)生理活性的影响作用。结果表明：干藻体在湿润的过程中,呼吸、光合和固氮活性依次恢复;且随水分含量的增加,光合活性和固氮活性逐渐增强,呼吸作用缓慢减弱并在一定水平上保持相对稳定,自由水是束缚水8倍左右时发菜生理活性全面恢复。吸水饱和的藻体在干燥过程中,光合、呼吸、固氮作用依次停止;呼吸作用随水分的丧失逐渐下降;固氮活性、光合活性在水分丧失20％～40％时有一定程度的增强,出现活性高峰;此后,生理活性下降,水分完全丧失时,光合作用终止,呼吸和固氮作用极其微弱。试验证明,水分是发状念珠藻生理活性的重要限制因子,适宜的水分有助于发菜维持正常的生理代谢和生长。     

Effects of UV-B Radiation and Periodic Desiccation on the Morphogenesis of the Edible Terrestrial Cyanobacterium Nostoc flagelliforme

The terrestrial cyanobacterium Nostoc flagelliforme Berk. et M. A. Curtis has been a popular food and herbal ingredient for hundreds of years. To meet great market demand and protect the local ecosystem, for decades researchers have tried to cultivate N. flagelliforme but have failed to get macroscopic filamentous thalli. In this study, single trichomes with 50 to 200 vegetative cells were induced from free-living cells by low light and used to investigate the morphogenesis of N. flagelliforme under low UV-B radiation and periodic desiccation. Low-fluence-rate UV-B (0.1 W m(-2)) did not inhibit trichome growth; however, it significantly increased the synthesis of extracellular polysaccharides and mycosporine-like amino acids and promoted sheath formation outside the trichomes. Under low UV-B radiation, single trichomes developed into filamentous thalli more than 1 cm long after 28 days of cultivation, most of which grew separately in liquid BG11 medium. With periodic desiccation treatment, the single trichomes formed flat or banded thalli that grew up to 2 cm long after 3 months on solid BG11 medium. When trichomes were cultivated on solid BG11 medium with alternate treatments of low UV-B and periodic desiccation, dark and scraggly filamentous thalli that grew up to about 3 cm in length after 40 days were obtained. In addition, the cultivation of trichomes on nitrogen-deficient solid BG11 medium (BG11(0)) suggested that nitrogen availability could affect the color and lubricity of newly developed thalli. This study provides promising techniques for artificial cultivation of N. flagelliforme in the future.     

发菜固氮基因nifD和nifK克隆及其失水条件下的差异表达

nifD和nifK编码钼铁固氮酶中的钼铁蛋白。为了解发菜nifD和nifK分子信息及对水分胁迫的响应机制,该研究设计了简并性引物克隆发菜nifD和nifK全长,进行原核表达和生物信息学分析,并对不同失水状态下发菜nifD和nifK在转录水平的差异表达和固氮酶活性的变化进行分析。结果表明,发菜nifD和nifK全长分别为1 443 bp和1 536 bp (登陆号为分别为KU886164和KU886165);将nifD和nifK在大肠杆菌中表达,分别获得一个约57 kD和58 kD的外源蛋白;生物信息学分析表明,nifD和nifK核苷酸序列和推译的氨基酸序列均与点形念珠藻(Nostoc punctiforme PCC 73102)高度一致性;nifD和nifK的二级结构主要有α-螺旋、β-折叠、β-转角和随机卷曲。此外,随着藻体含水量的逐渐降低,发菜nifD和nifK在转录水平上的表达量逐渐增加,但固氮酶活性呈现先增加后下降的趋势。研究结果为深入全面研究发菜固氮酶基因结构及其响应水分胁迫的固氮机理及氮代谢途径提供了基础。     

阳光紫外辐射对室内水培发状念珠藻生理特性的影响

发状念珠藻（Nostoc flagelliforme Bornet & Flahault）是一种重要的陆生经济蓝藻,室内培育出的原植体如何适应阳光辐射的问题尚需探讨。为此,作者将室内水培发菜置于阳光下培养,测定了其生长、有效光化学效率（F/Fm΄）和色素的变化。结果表明,较高的可见光（PAR,395－700 nm）和紫外辐射（UVR,280－395 nm）均导致水培发菜的F/Fm΄下降。第1天中午,PAR和UVR分别使F/Fm΄下降了54％和13％;傍晚,F/Fm΄有部分恢复。UVR对发菜适应阳光2d后的生长无负面作用。发菜在适应全阳光辐射期间,紫外吸收物质（Scytonemin和Mycosporine-like amino acids）含量不断增加,9d后,分别增加了124倍和9倍。这些紫外吸收物质的增加对发菜细胞降低光抑制,适应阳光辐射,起到了重要作用。本研究的结果可为水培发菜室外培养方法的建立提供一定的理论依据。     

低温电镜技术及其在植物（动物）材料研究中的应用

通过一些实例介绍了高压冷冻,冷冻置换和冷冻超薄切片等低温电镜样品制备技术,并且与传统方法对照,说明低温电镜技术的优越性,其中,发菜（Nostoc flagelliforme)营养细胞的冷冻超薄切片（未经化学固定,脱水）所显示的超微结构更客观地反映了生物样品的自然生理状态。此外,应用高压冷冻和冷冻置换的免疫标记电镜技术,首次对发菜营养细胞中的DNA进行定位,明确了核区的位置及范围。     

Oxidative stress in Nostoc flagelliforme subjected to desiccation and effects of exogenous oxidants on its photosynthetic recovery

The metabolism of reactive oxygen species in Nostoc flagelliforme and effects of exogenous oxidants on its photosynthetic recovery were investigated to obtain insight into oxidative stress in desiccation and its possible damaging impact on photosynthetic apparatus. No ascorbate was detected with ascorbate oxidase in N. flagelliforme. Superoxide dismutase (SOD) remained active even after three years drying storage and its activity was 78% of that in fully recovered samples. The SOD activity decreased during desiccation or in drying storage. Intracellular active oxygen production was studied by incubating samples in BG₁₁ medium for 2 h and measuring the oxidation of 2,7 -dichlorohydrofluorescein diacetate. The production rate was 38.11 nmol DCF_g (d.wt)^-1 h^-1 in dried field samples and was significantly higher than in fully recovered or air-dried samples. The balance between intracellular active oxygen production and the defense systems mightbreak down in air-dried and dried field samples. Treatment with exogenous oxidants slowed the photosynthetic recovery especially with singlet oxygen. Oxidative stress might play an important role in desiccationinduced damages to the photosynthetic apparatus.     

Colonization of wheat para-nodules by the N2-fixing cyanobacterium Nostoc sp. strain 2S9B

The treatment of wheat roots with 2,4-dichlorophenoxyacetic acid (2,4-D) led to the formation of tumour-like structures, para -nodules, which were readily colonized by cyanobacteria. The amount of cyanobacteria found on 2,4-D treated roots was 3.6 times higher than that found on untreated roots. Cyanobacteria penetrated the para - nodules by migrating in between loosely arranged cells that covered their surfaces or by penetrating the space at the junction of root and para -nodule. In plants treated with 2,4-D and co-cultivated with cyanobacteria in medium without combined nitrogen, the rate of acetylene reduction was three times that seen in untreated but colonized roots. Addition of 2,4-D itself did not change the rate of acetylene reduction in free-living culture of Nostoc sp. strain 2S9B. In plants treated with 2,4-D and co-cultivated with Nostoc sp. strain 2S9B in medium without combined nitrogen the nitrogen content of roots but not shoots was significantly increased.     

2种小球藻在鱼糜加工废水处理过程中的生长和油脂积累规律

以小球藻Chlorella pyrenoidsa F-9和C.vulgaris HYS-2为材料,研究小球藻在鱼糜加工废水中的生长速率、脂类积累规律和对氮磷营养的消除效果。结果表明,2种小球藻在鱼糜加工废水中快速生长,最高生物量达到0.293 g/L和0.276 g/L,分别是f/2对照培养基的2.4和1.6倍。小球藻HYS-2在鱼糜加工废水中油脂百分含量最高达到36.22%,为对照培养基的1.27倍,小球藻F-9在鱼糜加工废水中的油脂含量达到17.89%,为f/2培养基中的0.89倍。培养15 d后,2种小球藻对鱼糜加工废水中氮磷去除率均达90%以上。以上研究表明,用未经任何化学处理的鱼糜加工废水养殖小球藻在生长速率上远远大于f/2培养基,说明其具有较好地发展潜力,可以实现微藻的低成本养殖。