应用叶绿素荧光法测定微藻生物量的方法

为了探求叶绿素荧光值与海洋微藻生物量的关系,利用TD-700型叶绿素荧光仪测定了6种典型海洋赤潮藻在不同生长期(指数生长期和稳定生长期)时的叶绿素荧光值,同时用传统方法测定细胞密度,且利用Leica Qwin软件测量细胞大小。结果显示:微藻在同一生长时期细胞密度与叶绿素荧光值存在极显著的正相关关系,不同生长时期细胞密度也与单位叶绿素荧光值存在较显著正相关关系。因此,利用叶绿素荧光仪测定微藻生物量的方法是切实可行的,具有快捷方便、灵敏度高、可靠性强的优点。     

利用叶绿素荧光监测微藻的生长及生理状态

衣藻与扁藻是生产生物柴油的优势藻种,监测其生长和生理状况是利用它们生产生物柴油的关键环节。本研究通过叶绿素荧光对这两种微藻的细胞密度进行监测并利用多激发波长调制叶绿素荧光仪(MultiColor-PAM)检测其在不同生长条件下的生理状况,获得本研究中两种藻的适宜的培养温度/光照强度:衣藻为28℃/80μmol/m~2·s~(-1),扁藻为28℃/100μmol/m2·s~(-1),利用叶绿素荧光可以便捷准确的监测微藻的生长及生理状态,为微藻培养及其代谢产物积累的研究提供切实可行的无损伤的检测方法。     

邻苯二甲酸二丁酯对绿色巴夫藻的生长毒性和干扰效应

采用批次培养的方法研究了邻苯二甲酸二丁酯(DBP)对绿色巴夫藻(Pavlova viridis)的生长毒性和干扰效应。实验设置了0 mg·L^-1、2.5 mg·L^-1、3.0 mg·L^-1、3.5 mg·L^-1、4.0 mg·L^-1、5.0 mg·L^-1和7.5 mg·L^-1 共7个质量浓度梯度的DBP暴露处理组,测定了绿色巴夫藻的细胞密度、光合色素含量、叶绿素荧光特性及丙二醛(MDA)含量等指标,试图揭示DBP对绿色巴夫藻的生态毒性影响规律及机制。结果表明:DBP暴露对绿色巴夫藻的生长具有显著抑制作用,高质量浓度(5.0 mg·L^-1和7.5 mg·L^-1)DBP暴露下绿色巴夫藻细胞10 d内全部死亡;随着DBP暴露质量浓度增加,藻细胞叶绿素a和类胡萝卜素含量均显著降低;DBP暴露使光系统Ⅱ(PSⅡ)的最大光能转化效率(F_v/F_m)、潜在光化学活性(F_v/F_o)、实际光能转化效率(Yield)和光合电子传递速率(ETR)等指标也显著降低;DBP暴露还能够使绿色巴夫藻的细胞MDA含量显著增加。该研究进一步证实了DBP污染物对微藻光系统和酶类生理生化过程的干扰作用。     

海洋微藻四列藻对光周期改变的响应

以海洋微藻四列藻(Tetraselmis tetrathele)为试验材料,设置了短光周期(A)和长光周期(B)两组试验:A组分光周期1h、2h、4h、6h、8h和10h6个等级,B组分光周期14h、16h、18h、20h、22h和23h6个等级,均以光周期12h为对照,测定了四列藻的细胞密度、叶绿素a和蛋白质含量等指标.结果表明,在试验所设计的13个光周期等级中,光周期为6h、8h、10h、12h、14h、16h和18h等7个处理的藻细胞密度、叶绿素a和蛋白质含量等指标均达到较高水平.而光周期小于4h的3个处理和大于20h的3个处理,四列藻细胞的生长受到了明显抑制,细胞密度、叶绿素a和蛋白质含量均显著降低.     

锰浓度对米氏凯伦藻叶绿素荧光特性及生长的影响

运用实验生态学和叶绿素荧光分析技术,研究了锰浓度(10-12-10-4mol/L)对米氏凯伦藻(Karenia mikimotoi)叶绿素荧光特性及生长的影响。单因子方差分析结果表明:锰对米氏凯伦藻的生长和叶绿素荧光参数(Fv/Fm,Fv/Fo,ΦPSⅡ,ETR,qP,NPQ)均有显著影响(P0.05);米氏凯伦藻的叶绿素相对含量和细胞密度在10-12-10-8mol/L锰浓度间随着起始锰浓度的增大而增大,在10-8-10-4mol/L锰浓度间随锰浓度的增大而降低。多重比较结果表明,10-4mol/L锰浓度组的细胞密度和叶绿素相对含量显著低于其它处理组。锰浓度对荧光参数的影响与锰浓度范围和生长时期有关,当锰浓度为10-12-10-8mol/L时,荧光参数Fv/Fm,Fv/Fo,ΦPSⅡ,ETR在第3-9天随着起始锰浓度的增加而升高,Fv/Fm和Fv/Fo在第2-7天随培养时间的延长而增加。qP值在整个培养周期内随锰浓度升高呈下降趋势,各浓度组的NPQ则呈现先下降后上升趋势。相关性分析结果表明,从第3天开始至实验结束,10-4mol/L浓度组的叶绿素相对含量与细胞密度之间均呈极显著的正相关关系(P0.01)。荧光参数(Fv/Fm、Fv/Fo)、叶绿素相对含量以及细胞密度与锰浓度的相关性则随着锰浓度范围及培养天数的不同而变化。从第4天开始至培养结束,细胞密度、叶绿素相对含量均与锰浓度(10-8-10-4mol/L)呈极显著的负相关(P0.01)。探讨了叶绿素荧光技术在赤潮藻研究中的应用。     

不同培养基对发菜细胞生长和光合活性的影响

研究测定了发菜(NostocflagelliformeBorn.etFlah.)细胞在不同培养基中的生长速率、光合作用和叶绿素荧光活性。结果显示培养11d后:Detmer培养基中叶绿素a的含量为1.08mg/L,Kratz-Myers培养基中叶绿素a的含量为1.87mg/L,水生104号培养基中叶绿素a的含量为1.21mg/L,BG11培养基中叶绿素a的含量为2.18mg/L,表明在BG11培养基中培养的细胞具有最高生长速率;与另外4种不同浓度的BG11培养基相比,上述BG11培养基培养的发菜具有最大的光合速率Pm(218.1μmolO2.mg-1chla.h-1)和最高的PSII光化学效率(Fv/Fm=0.349)。实验结果表明,BG11是适合发菜生长的培养基,对其光合作用和叶绿素荧光活性具有显著促进作用。     

两种生境条件下6种牧草叶绿素含量及荧光参数的比较

昆仑山前山牧场海拔较高, 策勒绿洲海拔相对较低, 两者生境差异较大。以昆仑山前山牧场和策勒绿洲边缘两种不同生境条件下生长的6种牧草: 冰草(Agropyron cristatum)、无芒雀麦(Bromus inermis)、矮生高羊茅(Festuca elata)、披碱草(Elymus dahuricus )、红豆草(Onobrychis pulchella)及和田大叶(Medicago sativa var. luxurians)为试验材料, 研究了不同生境条件下牧草叶片叶绿素含量及叶绿素荧光动力学参数的变化情况。结果显示: (1)在两种生境条件下, 昆仑山前山牧场生境生长的牧草叶绿素a、叶绿素b、总叶绿素的含量明显较高, 生长在策勒绿洲生境的牧草品种叶绿素a/b值较高; (2)昆仑山前山牧场生境牧草最大荧光、光系统II (PSII)最大光化学效率、PSII潜在活性和单位面积反应中心的数量的值明显高于策勒绿洲生境品种, 而初始荧光、单位反应中心吸收的光能、单位反应中心捕获的能量、单位反应中心耗散的能量、荧光诱导曲线初始斜率值则低于策勒绿洲生境品种。因此, 两种生境下环境因子发生了改变, 对牧草产生综合的胁迫作用; 策勒绿洲生境明显对牧草生长产生了抑制, 策勒绿洲生境牧草的色素含量降低以及PSII的机构遭到损坏, 导致反应中心一部分失活或裂解, 剩余有活性的反应中心的效率增加, 昆仑山生境则相对比较适宜牧草生长; 两种生境不同牧草叶绿素含量和叶绿素荧光参数的变化幅度不同。     

VB1、VB12和VH对雨生红球藻生长及类胡萝卜素含量的影响

以淡水经济微藻雨生红球藻(Haematococcus pluvialis CH-1)为试验藻种,分别添加维生素B₁(VB₁)、B₁₂(VB₁₂)和维生素H(VH),每种维生素各分6个浓度梯度,测定了雨生红球藻的细胞密度、生物量、叶绿素a和类胡萝卜素含量等指标。结果表明,分别添加维生素B₁、B₁₂和H均显著促进了雨生红球藻细胞的生长。维生素B₁、B₁₂和H对雨生红球藻的最佳添加浓度分别为10mg·L^-1、50μg·L^-1和500μg·L^-1。在维生素B₁、B₁₂和H各自的最佳浓度处理下,雨生红球藻的细胞密度、生物量、叶绿素a和类胡萝卜素含量等4项指标均比对照有显著提高:维生素B₁处理分别提高了20.1%、14.6%、23.2%和21.3%;维生素B₁₂处理分别提高了29.5%、30.0%、28.0%和24.4%;维生素H处理分别提高了17.1%、29.2%、21.8%和10.1%。在雨生红球藻规模化生产的游动细胞培养阶段,适当地添加维生素B₁、B₁₂和H均能够有效地提高藻细胞密度、生物量、叶绿素a和类胡萝卜素含量。     

葛仙米生长的尺度效应

为考察葛仙米(Nostoc sphaeroides)群体粒径变化对营养成分和光合活性的影响, 测定了生长过程中不同粒径群体的色素成分、光合特性和多糖含量。结果表明: 叶绿素a、藻胆蛋白含量随群体粒径增大而降低; 类胡萝卜素含量在粒径4.85 mm时最高, 藻蓝蛋白/叶绿素a、藻红蛋白/叶绿素a以及别藻蓝蛋白/叶绿素a的比值随粒径增大先降低后升高; 最大潜在光合能力(F_v/F_m)随群体粒径增大逐渐降低, 大群体可通过光系统I介导的环式电子传递链耗散多余能量以保护光合作用系统; 多糖含量在粒径3.03 mm时最高。研究结果显示葛仙米生长具有明显的尺度效应, 细胞组分与光合活性对群体粒径变化具有显著的响应, 开展规模化培养和采收需要关注这一效应。     

蓝藻水华水样总氮含量的测定

为评估蓝藻水华藻密度对水样中总氮含量测定的影响,采用实验室培养纯藻和天然微囊藻水华样品,利用碱性过硫酸钾消解紫外分光光度法,测定了不同藻密度水样TN的含量并进行了比较分析。研究发现,如果藻密度较高的水样稀释不充分, TN值会被严重低估。对太湖水华水样分析结果表明,叶绿素a与TN的相应值平均为0.156 mg N/μg Chl. a,干藻样的氮含量平均为16%。这些数据有助于估算蓝藻水华对水体TN的贡献。     

微藻无菌化技术的研究进展

无菌纯藻是深入开展藻类生理学和遗传学研究的基础。目前已有涂布划线法,离心洗涤技术、稀释滤过技术、辐照技术、毛细吸管技术、抗生素技术、化学消毒技术、利用其他生理特性等技术用于微藻的纯化。拟介绍国内外近年来微藻无菌纯化技术的研究进展。     

Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence

A rapid estimation method of the intracellular lipid content in microorganisms using a fluorescent probe, Nile red, was established by optimization of the Nile red staining and data processing. The protocol was designed to be applicable to a wide range of microorganisms and culture conditions. In the optimized procedure, cells diluted with buffer were stained with 0.24-0.47 microg/ml of Nile red for 5 min, and the fluorescent emission spectra in the wavelength region of 400 to 700 nm excited at 488 nm were acquired before and after the Nile red addition. The fluorescence intensity corresponding to the intracellular lipid amount was determined at the peak of the corrected spectrum. The value showed a linear relation with the lipid content of various oleaginous fungi and yeasts measured by the conventional method. The relative intensities against the unit lipid amounts were almost similar except for one yeast. For the application to mycelia forming various types of pellets, a simple and easy pretreatment of shaking with glass beads for 5-10 min was added to the protocol. The established method was applicable to estimate the lipid content of a wide range of microorganism cultures containing 2-5000 microg-lipid/ml-broth.     

Light-dependent,but phytochrome-independent,translational control of the accumulation of the P700 chlorophyll-a protein of photosystem I in barley (Hordeum vulgare L.)

This work reports on the regulation of synthesis of the P700 chlorophyll-a apoprotein of photosystem I in barley. The mRNA for the P700 apoprotein is almost exclusively confined to the plastid membrane-bound polysomes. However, the mRNA for the 32-kDa herbicide-binding protein of photosystem II is found in both the soluble and membrane-bound polysomes.The mRNA for the P700 apoprotein is found in similar amounts in dark-grown and light-grown wild-type as well as mutant xantha-l⁸¹ barley. The latter mutant is deficient in chlorophyll biosynthesis. However, while wild-type leaves accumulate the P700 chlorophyll-a protein only in the light, mutant leaves never accumulate the P700 apoprotein.A more sensitive approach was taken using isolated plastids to study P700 apoprotein synthesis. Etioplasts did not synthesize detectable P700 apoprotein even when the etioplasts were exposed to light. However, only a 1-min exposure of leaves to light was necessary to induce P700 apoprotein synthesis by isolated plastids.Phytochrome involvement in controlling P700 apoprotein synthesis was tested by using red/farred light treatment of leaves. These treatments showed no far-red reversibility of red-induced P700-apoprotein synthesis in isolated plastids even after 3 h of darkness after the light treatments. From these data we conclude that the accumulation of P700 apopootein is not under the control of phytochrome and that the light induction of P700 apoprotein is most likely mediated through the protochlorophyllide/chlorophyllide system. This control, however, may also involve cytoplasmic signals as the synthesis of the P700 apoprotein is not turned on in illuminated etioplasts.     

枯草芽孢杆菌fmbJ产生的新型抗微生物物质体外抗NDV和IBDV的活性分析

测定了枯草芽孢杆菌fmbJ株产生的新型抗微生物物质的体外抗新城疫病毒(Newcastle disease virus,NDV)lasota株、传染性法式囊病病毒(Infectious Bursal Disease Virus,IBDV)哈尔滨(H)株作用。结果表明该新型抗微生物物质对鸡胚成纤维(Chicken Embryo Fibroblasts,CEF)细胞的TD50和TD0分别为128.95mg/L、25.79mg/L;对NDVlasota株、IBDV H株所致细胞病变效应有明显的抑制作用,可使细胞存活率显著升高;该抗微生物物质具有抗NDVlasota株、IBDV H株作用;并具有预防其感染及抑制其复制的作用。其抗病毒作用效果和病毒唑相当,由于其对CEF细胞的毒性较弱,可作为一种抗病毒药物进行开发研究。     

Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate

Armed antibody-based targeted molecular therapies offer the possibility of effective tumor control with a minimum of side effects. Photoimmunotherapy (PIT) employs a monoclonal antibody-phototoxic phthalocyanine dye, IR700 conjugate, that is activated by focal near-infrared (NIR) light irradiation after antibody binding to the targeted tumor cell surface leading to rapid necrotic cell death. Therapy by single NIR light irradiation was effective without significant side effects; however, recurrences were seen in most treated mice probably because of inhomogeneous distribution of panitumumab-IR700 immunoconjugate in the tumor, leading to ineffective PIT. We describe here an optimized regimen of effective PIT method for the same HER1-overexpressing tumor model (A431) with fractionated administration of panitumumab-IR700 conjugate followed by systematic repeated NIR light irradiation to the tumor based on timing of antibody redistribution into the remnant tumor under the guidance of IR700 fluorescence signal. Eighty percent of the A431 tumors were eradicated with repeated PIT without apparent side effects and survived tumor-free for more than 120 days even after stopping therapy at day 30. Therapeutic effects were monitored using IR700 fluorescent signal. PIT is a promising highly selective and clinically feasible theranostic method for treatment of mAb-binding tumors with minimal off-target effects.     

稻虾共作水体浮游植物群落结构特征分析

为认识稻虾共作水体浮游植物群落结构特征, 于2016年4月至12月对江汉平原4处稻虾共作水体浮游植物和理化因子开展了逐月调查与分析。共鉴定出浮游植物7门124种, 其中绿藻78种、蓝藻16种、硅藻15种、裸藻3种、隐藻2种、甲藻7种、金藻3种。6—9月浮游植物的种类数、细胞密度、叶绿素a含量达到最大值, 最小值出现在12月份; 浮游植物细胞密度波动范围在1.37×105—2.93×108 cells/L, 叶绿素a含量的变化范围为0.15—208.60 μg/L。调查期间浮游植物的优势种共28种, 主要优势种有颤藻、蓝纤维藻、微囊藻、小球藻、隐藻等。浮游植物Shannon-Wiener多样性指数周年变化范围为0.64—6.3, 多样性指数最高出现在10月份, 最低出现在8月份。结果显示, 稻虾共作水体浮游植物群落结构较复杂, 细胞密度变化显示时空的一致性, 优势种组成以及优势度存在明显的空间差异(P<0.05), 稻田的浅水环境以及小龙虾的养殖行为显著影响浮游植物群落的结构。鉴于藻类作为初级生产者对于水生态环境和小龙虾健康具有重要作用, 关注种养结合水体中藻类群落的演变规律对于保障稻田种养的综合效益具有积极意义。     

The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.     

微藻对常用抗生素敏感性的研究进展

在微藻培养过程中,需在藻液中加入抗生素以达到除菌、抑菌的目的。对近年来微藻的抗生素纯化技术进行了综述,对选择抗生素的方法、抗生素抑菌机理及常用抗生素对微藻培养的影响等方面进行了归纳和分析,并对无菌化培养的研究前景做了展望。     

Colocalization of TD-60 and INCENP throughout G2 and mitosis: evidence for their possible interaction in signalling cytokinesis

TD-60 and INCENP are two members of the chromosome passenger protein family, and each has been suggested to play a role in the control of cytokinesis. Here we demonstrate by confocal immunofluorescence microscopy that TD-60 and INCENP distribute identically throughout the cell cycle. Both appear coordinately in G2-phase nuclei and become concentrated at centromeres during prophase. TD-60 and INCENP both then leave the chromosome together during anaphase and redistribute to the spindle midzone, as do other chromosome passenger proteins, and traverse the entire equatorial diameter from cortex to cortex. By image overlay and pixel count analysis we show that TD-60 and INCENP are distinct among known chromosome passenger proteins in extending beyond the spindle to the cortex. Further, we show that the cytokinesis-associated protein kinase AIM-1 also shares this distribution property. We suggest that this redistribution is required to signal cytokinesis. TD-60 and INCENP also show identical localization in cells that exit mitosis in the presence of dihydrocytochalasin B (DCB), an inhibitor of actin assembly. Such cells can resume cleavage upon removal of DCB and in a recovery subpopulation that cleaves only on one side, these proteins both colocalize to the cortex only where a cleavage furrow forms. Given the coincident distribution of TD-60 and INCENP during both interphase and mitosis, we suggest that these proteins may cooperate, perhaps within a protein complex, in signalling cytokinesis. Such a mechanism, using chromosome passenger proteins, may ensure that cytokinesis occurs only between the separated chromatids, and only after they have segregated. Received: 12 August 1998; in revised form: 1 September 1998 / Accepted: 2 September 1998