发状念珠藻光合特征及其在复水－失水过程中能量传递变化的研究

摘要 "发状念珠藻（Nostoc flagelliforme Born. Et Flah.），俗名发菜，是生长于干旱、半干旱土壤表面的陆生蓝藻，具有极强的抗旱能力。发菜光合作用方面的研究大多处于整体细胞水平，且研究手段非常有限。本实验对发菜光合特征进行深入研究，并探讨了发菜在干湿交替过程中能量传递的变化情况。 叶绿素和藻胆素是发菜细胞中两种主要的光合色素。发菜复水后光合活性完全恢复时，在室温（20℃）或低温（77K）下，其绝大部分的荧光是由于藻胆素被激发而产生。在室温下，大部分荧光来自藻胆体;当叶绿素被激发后，产生的荧光非常微弱。在低温下，藻胆素被激发后，荧光发射光谱中可分辨出藻胆蛋白、光系统Ⅰ和光系统Ⅱ的发射峰;叶绿素被激发后，荧光发射光谱包括光系统Ⅰ和光系统Ⅱ的荧光。相比之下，室温荧光发射光谱不适于用做发菜细胞光合作用方面的研究。 我们设计了一种新方法，从野生发菜细胞中分离得到类囊体膜及细胞质膜，并对其性质进行分析。发菜细胞外复杂的胶质结构使得现有破碎其它蓝藻细胞的方法无法破碎发菜细胞。通过实验发现，联合使用细胞破碎仪和毛地黄皂甙（0.3%）可有效破碎发菜细胞;并且毛地黄皂甙在低浓度下（≦0.5%），对色素与蛋白的结合不会造成破坏作用。随后，通过蔗糖密度梯度离心可将细胞质膜与类囊体膜分离。发菜类囊体膜的光谱性质与其它蓝藻相似。细胞质膜除结合有类胡萝卜素外，还结合有少量叶绿素前体。类囊体膜和细胞质膜膜脂及脂肪酸组成相似。其中，十六碳烯酸[16：1(9)]和亚麻酸[18：3(9,12,15)]是含量最高的两种脂肪酸，分别占总脂肪酸含量的三分之一左右。高含量的多不饱和脂肪酸可能和发菜极强的抗旱能力有关。 我们首次对发菜捕光色素蛋白复合物－藻胆体的组成和结构进行分析。发菜藻胆体为“3核＋6杆”的半圆盘结构。组成藻胆体的藻胆蛋白包括藻蓝蛋白和别藻蓝蛋白。两个藻蓝蛋白六聚体通过连接肽组成藻胆体的“杆”结构。在“杆”结构中等量分布着两条连接肽（分子量分别为29kDa和34kDa）为杆连接肽和核杆连接肽。而“核”结构中核膜连接肽的分子量为103kDa。 发菜在无霜期，几乎每天经历一次复水－干燥过程：夜间的结露使发菜在黑暗中复水，而清晨太阳升起后，在光照下迅速失水进入休眠状态。我们研究了发菜在黑暗中的复水过程及在光照下失水过程中藻胆体与光系统能量传递的变化情况。发菜在黑暗中复水后，光系统Ⅱ活性无法恢复。藻胆体内及藻胆体与光系统Ⅰ的能量传递在5分钟内恢复;而藻胆体与光系统Ⅱ的能量传递只能部分恢复。我们设想，发菜在复水过程中通过双扳机－水和光－控制光合活性的恢复，以及在黑暗中部分恢复藻胆体与光系统Ⅱ的能量传递，将减少不必要的能量消耗与通过光合作用储备尽可能多的化学能－这两个生存策略有机的结合起来。发菜在光照下的失水过程中，光合活性在含水量降至90％前基本保持稳定，随后迅速下降。而在含水量达到150％后，藻胆体内的能量传递便开始受到抑制，并且随着含水量的降低，该抑制现象逐步加剧。这样，发菜在干燥过程中，通过抑制藻胆体内的能量传递，减少了传递到光系统Ⅱ反应中心的能量，从而避免了在光合活性下降过程中过剩光能对光系统Ⅱ产生的破坏作用。"

Effects of gamma-aminobutyric acid, progesterone and ionophore A23187 on acrosome reaction of tree shrew sperm in vitro: examination of acrosome reaction with an improved fluorescence microscopy

A number of acrosome reaction (AR) initiators have been found to be effective in inducing AR of human, laboratory and domestic animal sperm. Using an improved simple fluorescence microscopy, effects of gamma-aminobutyric acid (GABA), progesterone and ionophore A23187 on sperm AR of tree shrew, a useful animal model in biomedical research, have been investigated. Spontaneous AR in 4.92-7.53% of viable sperm was observed. Complete AR in 10.31-18.25% of viable tree shrew sperm was obviously induced by 5 mu M and 10 mu M calcium ionophore A23187, 1 mM GABA, and 5 mu M progesterone, and there were no significant differences between their abilities to initiate complete AR. No significant differences of AR percentages between 1- and 2-h treatments with A23187, progesterone and/or GABA were observed. These results suggested that the responses of tree shrew sperm to these AR initiators are similar to that of human and other mammalian sperm. (C) 1997 Elsevier Science B.V.

EFFECT OF ELEVATED BICARBONATE CONCENTRATION ON GROWTH, CHLOROPHYLL A FLUORESCENCE AND ULTRA-STRUCTURE OF Microcystis aeruginosa (CYANOBACTERIUM)

The influence of bicarbonate (HCO3-) on Microcystis aeruginosa FACHB 905 was assessed in this study. Growth curves, chlorophyll a fluorescence and ultrastructure were measured at two HCO3- concentrations, 2.3 mM and 12.4 mM. A treatment of sodium chloride (NaCl) was also conducted alongside to establish the influence level of sodium. It was found that upon treatment with elevated HCO3- concentrations of 2.3 mM and 12.4 mM, cell densities were 13% and 27% (respectively) higher than controls. In photosynthetic performance, elevated HCO3- concentration initially stimulated Fv/Fm at the prophase of culture and then subsequently inhibited it. The inhibition of 2.3mM was higher than that of 12.4mM HCO3-. The maximum relative electron transport rate (ETRmax) exhibited inhibition at elevated HCO3- concentrations. DI0/CS was decreased at 2.3 mM and increased at 12.4mM. In the case of both treatments. ABS/CSI TR0/CS, ET0/CS, RC/CS0 and RC/CSm were decreased by elevated HCO3- concentrations, which indicated damage to photosynthetic apparati and an inactivation of a fraction of reaction centers. This point was also proven by ultrastructural photos. High HCO3--exposed cells lost the characteristic photosynthetic membrane arrangement compared with the control and high salinity treated samples. At the 2.3mM concentration of HCO3-. damage to photosynthetic apparati caused decreased photosynthetic activity. These findings suggested that elevated HCO3- concentration stimulated the growth and photosynthesis of M. aeruginosa FACHB 905 in a short time. Exposure to high HCO3- concentrations for a longer period of time will damage photosynthetic apparatus. In addition, the ultrastructure indicated that elevated HCO3--concentration lead to photosynthetic apparati damage. In our experiment, it was observed that the inhibition effect of 2.3mM HCO3- was higher than that of 12.4mM HCO3-. We hypothesized that M. aeruginosa FACHB 905 induced a protective mechanism under high concentrations of HCO3-.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

Effects of sand burial on biomass, chlorophyll fluorescence and extracellular polysaccharides of man-made cyanobacterial crusts under experimental conditions

Soil cyanobacterial crusts occur throughout the world, especially in the semiarid and arid regions. It always encounters sand burial, which is an important feature of mobile sand dunes. A greenhouse 41 study was conducted to determine the effects of sand burial on biomass, chlorophyll fluorescence and extracellular polysaccharides of man-made cyanobacterial crusts in six periods of time (0, 5, 10, 15, 20 and 30 d after burying) and at five depths (0, 0.2, 0.5, 1 and 2cm). The results indicated that with the increase of the burial time and burial depth extracellular polysaccharides content and Fv/Fm decreased correspondingly and there were no significant differences between 20 and 30 burial days under different burial depths. The degradation of chlorophyll a content appeared only at 20 and 30 burial days and there was also no significant difference between them under different burial depths. It was also observed a simultaneous decrease of the values of the Fv/Fm and the content of extracellular polysaccharides happened in the crusted cyanobacterium Microcoleus vaginatus Gom. It may suggest that there exists a relationship between extracellular polysaccharides and recovery of the activity of photosystem II (PS II) after rehydration.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Study on fluorescence emission and synchronous-scan fluorescence spectrea of Nitzschia hantzschiana solution with FE(III)

The characterization of the algal Nitzschia hantzschiana solution with (or without) Fe(III) was carried out using fluorescence emission and synchronous-scan spectroscopy. An emission peak (excited at 440 nm) was observed at 675 nm for Nitzschia hantzschiana solution. The effective characterization method used was synchronous-scan fluorescence spectroscopy (SFS). A wavelength difference (Delta lambda) of 90 nm was maintained between excitation and emission wavelengths. The peak was observed at about 236(ex) nm (326(em) nm) for synchronous fluorescence spectroscopy. Fe(III) was an effective quencher. The relationship between I-0/I (quenching efficiency) and c (concentration of Fe (III) added) was a linear correlation for the algal solution with Fe(III). Effects of pH on synchronous-scan fluorescence intensity were evident.

Preparation of pH-sensitive polymer by thermal initiation polymerization and its application in fluorescence immunoassay

A fluorescence immunoassay for human IgG (Ag) was developed using a pH-sensitive polymer prepared by thermal initiation or redox initiation polymerization as a carrier. In the competitive immunoassay, appropriate quantity of Ag was immobilized on the polymer and the standard Ag (or sample) solution, and a constant amount of fluorescein isothiocyanate labeled goat anti-human IgG antibody (Ab-FITC) was added. Immobilized Ag and the standard (or sample) Ag competed for binding to the Ab-FITC in 37 C in homogeneous format. After changing the pH to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. The results showed that the immobilization efficiency, immunological reaction activities of immobilized Au and phase transition pH range were improved as Ag was immobilized by thermal initiation instead of redox initiation polymerization. Under optimum conditions, the calibration graphs for the Ag in both methods, thermal initiation and redox initiation, were linear over the concentration range of 0.0-1000 ng mL(-1), with detection limits 8 (thermal initiation) and 12 ng mL(1) (redox initiation), respectively. Moreover, some pH-sensitive polymer prepared only in organic solvent or under high temperature could also be used as an immunoreaction carrier by thermal initiation polymerization. Thermal initiation polymerization was a better immobilization mode. (C) 2004 Elsevier B.V. All rights reserved.