水稻灌浆期籽粒与 胚乳发育的蛋白质组学分析

籽粒的灌浆是将光合器官合成的有机物贮存在籽粒中的过程。这一过程直接决定了籽粒的产量及品质。先前研究表明灌浆籽粒中贮存物质的累积是各种代谢活动和细胞学过程协同作用的结果，但灌浆的分子机制目前还不是非常清楚。水稻是研究籽粒灌浆的优良模式材料，不仅因为它是世界上最重要的淀粉食物来源，更重要的是其全基因组的测序完成为分子机制的研究带来极大的便利。我们对发育水稻籽粒的观察表明在开花后6 天，籽粒就已完成了胚的分化和胚乳的细胞化；此后籽粒经历了一个显著的细胞增大过程，并在开花后12 天左右达到成熟籽粒的大小；而籽粒的灌浆过程起始于开花后6 天，这个过程一直持续到开花后20 天。因此，我们将开花后6 天到20 天的籽粒分为8 个连续的发育阶段进行动态的蛋白质组分析，396个蛋白点的表达在灌浆过程中发生了两倍以上变化。质谱鉴定得到的345 个差异表达的蛋白划分为10 个不同的功能类别。其中新陈代谢类（45%）和蛋白合成/终点（destination）类（20%）两个功能类别中就包括了大多数的差异表达蛋白，预示着这两类蛋白在籽粒发育中的重要性。蛋白功能群的表达分析显示与淀粉合成和乙醇发酵相关的蛋白在发育过程中大幅度的上调，而与碳代谢中心过程（糖酵解和三羧酸循环）相关蛋白呈现明显的下调趋势。大多数的功能类或（亚类）也呈现出下调的表达趋势，如细胞生长/分裂类，蛋白合成类，水解类，信号传导类和转录类。蛋白表达分析的结果表明蛋白的表达随籽粒的发育发生了显著的变化，这些变化与籽粒在不同阶段的发育和代谢过程密切相关并协调一致，是细胞从生长分裂过渡到以淀粉合成为中心的物质基础。同时也说明代谢重点由中心碳代谢向乙醇发酵的转变对于籽粒的发育和淀粉的合成与累积具有重要意义。 籽粒发育的研究表明在长到成熟籽粒大小后（开花后12 天），籽粒的代谢集中到淀粉累积途径上，一直持续到进入脱水期（18 天），绝大多数淀粉合成相关蛋白在这期间到达表达的顶点。为了解淀粉累积关键时期淀粉合成关键部位（胚乳）的发育规律，我们进一步应用DIGE 技术对这一淀粉累积关键时期（灌浆中后期，开花后12 到18 天）的蛋白表达特性进行分析。细胞学的观察发现胚乳在灌浆后期先后经历了过氧化氢的爆发、半透明胚乳的形成以及胚乳细胞死亡事件。相应的DIGE 分析显示有321 个蛋白点在胚乳的后期发育中发生了显著的表达变化。细胞学的观察结合DIGE 分析显示胚乳的后期发育是一个典型的衰老过程：细胞结构的崩溃；氧化自由基的爆发；脱水干燥；蛋白、脂类和DNA 由同化作用向异化作用的代谢转化。与代谢转化相伴随的细胞营养的重新分配是胚乳后期发育的一个显著过程。DIGE分析全面展示了参与营养重新分配相关蛋白在后期发育中的表达变化，为细胞学中观察到的有机物向淀粉的转化提供了清晰的蛋白水平的证据支持。在鉴定的差异表达蛋白中有2/3 的蛋白是已知的对氧化电位变化敏感的蛋白，表明由H2O2 爆发形成的氧化压力将引起氧化还原调控从而对胚乳的后期发育进行全面的影响。而其中与碳元素代谢相关的代谢途径中尤其富含氧化还原电位敏感的蛋白，表明后期的营养重新分配以及淀粉的累积受到氧化还原电位的紧密调控。另一方面，H2O2 的爆发激发了胚乳中的抗氧化体系。由抗氧化蛋白（如thioredoxin、抗坏血酸和超氧化物歧化酶等）、氧化还原敏感蛋白、代谢中间产物以及glyoxalase 构成的抗氧化体系在胚乳后期发育中协同作用调节氧化还原电位的变化，从而控制胚乳细胞衰老的节奏。另外，我们发现与RraA 相关的转录本的调控在胚乳发育末期急剧上调，在调控的代谢途径、调控时间以及调控的部位与氧化还原调控相重叠，并且支持RraA 活动有利于胚乳细胞对氧化压力的适应。所有这些结果表明内生的过氧化氢（或氧化自由基）在胚乳的后期发育和淀粉累积中起到核心的调控作用。

Direct electron transfer to cytochrome c oxidase in self-assembled monolayers on gold electrodes

The monolayer of cytochrome c oxidase maintaining physiological activity and attached covalently to the self-assembled monolayers of 3-mercaptopropionic acid (MPA) on a gold electrode was obtained. The results of cyclic voltammetry show that direct electron transfer between cytochrome c oxidase and the electrode surface is a fast and diffusionless process. MPA has a dual role as both electrode modifier and the bridging molecule which: keeps cytochrome c oxidase at an appropriate orientation without denaturation and enables direct electron transfer between the protein and the modified electrode. Immobilized cytochrome c oxidase exhibits biphasic phenomena between the concentration of the electrolyte and the normal potentials; meanwhile its electrochemical behavior is also influenced by the buffer components. The quasi-reversible electron transfer process of cytochrome c oxidase with formal potential 385 mV vs. SHE in 5mM phosphate buffer solution (pH 6.4) corresponds to the redox reaction of cyt a(3) in cytochrome c oxidase, and the heterogeneous electron transfer rate constant obtained is 1.56 s(-1). By cyclic voltammetry measurements, it was observed that oxidation and reduction of cytochrome c in solution were catalyzed by the immobilized cytochrome c oxidase. This cytochrome c oxidase/MPA/Au system provides a good mimetic model to study the physiological functions of membrane-associated enzymes and hopefully to build a third-generation biosensor without using a mediator.

Electrochemical study of the interfacial characteristics of redox-active viologen thiol self-assembled monolayers

The electrochemical behavior of the electroactive self-assembled monolayers (SAMs) of thiol-functionalized viologen, CH3(CH2)(9)V2+(CH2)(8)SH, where V2+ is a viologen group, on the gold electrodes is examined by cyclic voltammetry and electrochemical a.c. impedance. A monolayer of viologen is immobilized on the gold electrode surface via the Au-S bond and the normal potentials corresponding to the two successive one-electron transfer processes of the viologen active centers are -310 mV and -652 mV (vs. Ag/AgCl) in 0.1 mol l(-1) phosphate buffer solution (pH 6.96) respectively. These results suggest that the viologen SAMs are stable and well-behaved monolayers. The experimental impedance data corresponding to different forms of viologen group have been fitted to equivalent electrical circuits, and the surface capacitances and resistances have been given. The heterogenous electron transfer rates of the first and the second redox processes are 7.57 s(-1) and 1.49 s(-1) respectively through a.c. impedance.

EST-based identification of genes expressed in the hypothalamus of male orange-spotted grouper (Epinephelus coioides)

The orange-spotted grouper, Epinephelus coioides, is an important marine aquaculture fish, but its large-scale aquaculture has been hindered by the rarity of natural males because it is a protogynous hermaphroditic fish. Hypothalamus-pituitary-gonad is an important endocrine axis in regulating reproduction and sex differentiation. To reveal the molecular mechanism of hypothalamic physiological functions, we performed he studies on identification of genes expressed in the hypothalamus of male orange-spotted grouper using EST and RT-PCR strategy. A total of 1006 ESTs were sequenced, and 402 (39.96%) clones were identified as known genes and 604 (60.04%) as unknown genes. The 402 clones of known gene products represent transcripts of 18 1 genes. Moreover, the expression patterns of 26 unknown genes were analyzed in various tissues, such as liver, kidney, spleen, fat, heart, muscle, pituitary, hypothalamus, telencephalon, cerebellum, midbrain, medulla oblongata, ovary and testes. Five different categories of expression patterns were observed from them. Several unknown ESTs, such as DN551996, DN551998, DN552082, and DN552070, were detected to be hypothalamus-specific, brains-specific, or hypothalamus and gonad-specific genes. Interestingly, DN551996, not only exhibiting expression differences between ovary and testis, but also showing sex-dependent differences in hypothalamus of grouper, might play significant role in grouper reproduction or sex inversion. Further functional studies on these genes will provide more information on molecule regulation mechanism of sex inversion in groupers. (c) 2006 Elsevier B.V All fights reserved.

Tetrahydrothiophenium-based ionic liquids for high efficiency dye-sensitized solar cells

Binary melts of S-ethyltetrahydrothiophenium iodide and dicyanoamide (or tricyanomethide) have been employed for dye-sensitized solar cells with high power conversion efficiencies up to 6.9% under the illumination of AM 1.5G full sunlight. We have further shown that the transport of triiodide in ionic liquids with high iodide concentration is viscosity-dependent in terms of a physical diffusion coupled bond exchange mechanism apart from the simple physical diffusion.

Amperometric glucose biosensor based on single-walled carbon nanohorns

The biosensing application of single-walled carbon nanohorns (SWCNHs) was demonstrated through fabrication of an amperometric glucose biosensor. The biosensor was constructed by encapsulating glucose oxidase in the Nafion-SWCNHs composite film. The cyclic voltammograms for glucose oxidase immobilized on the composite film displayed a pair of well-defined and nearly symmetric redox peaks with a formal potential of -0.453V. The biosensor had good electrocatalytic activity toward oxidation of glucose.

Hydrogen peroxide biosensor based on direct electrochemistry of soybean Peroxidase immobilized on single-walled carbon nanohorn modified electrode

Single-walled carbon nanohorns (SWCNHs) were used as a novel and biocompatible matrix for fabricating biosensing devices. The direct immobilization of acid-stable and thermostable soybean peroxidase (SBP) on SWCNH modified electrode surface can realize the direct electrochemistry of enzyme. Cyclic voltammogram of the adsorbed SBP displays a pair of redox peaks with a formal potential of -0.24V in pH 5 phosphate buffer solution.

Sensitive detection of cysteine based on fluorescent silver clusters

In this work,we report the application of novel, water-soluble fluorescent Ag clusters in fluorescent sensors for detecting cysteine, an important biological analyte. The fluorescence of poly(methacrylic acid) (PMAA)templated Ag clusters was found to be quenched effectively by cysteine, but not when the other alpha-amino acids were present. By virtue of the specific response, a new, simple, and sensitive fluorescent method for detecting cysteine has been developed based on Ag clusters. The present assay allows for the selective determination of cysteine in the range of 2.5 x 10(-8) to 6.0 x 10(-6) M with a detection limit of 20 nM at a signal-to-noise ratio of 3. Based on the absorption and fluorescence studies, we suggested that cysteine quenched the emission by the thiol-adsorption-accelerated oxidation of the emissive Ag clusters. The present study shows a promising step toward the application of silver clusters, a new class of attractive fluorescence probes.

Gold nanoparticle-based near-infrared fluorescent detection of biological thiols in human plasma

In this work, a new fluorescent method for sensitive detection of biological thiols in human plasma was developed using a near-infrared (NIR) fluorescent dye, FR 730. The sensing approach was based on the strong affinity of thiols to gold and highly efficient fluorescent quenching ability of gold nanoparticles (Au NPs). In the presence of thiols, the NIR fluorescence would enhance dramatically due to desorption of FR 730 from the surfaces of Au NPs, which allowed the analysis of thiol-containing amino acids in a very simple approach. The size of Au NPs was found to affect the fluorescent assay and the best response for cysteine detection was achieved when using Au NPs with the diameter of 24 nm, where a linear range of 2.5 x 10(-8) M to 4.0 x 10(-6) M and a detection limit of as low as 10 nM was obtained. This method also demonstrated a high selectivity to thiol-containing amino acids due to the strong affinity of thiols to gold.

Sensitive and Selective Sensor for Biothiols in the Cell Based on the Recovered Fluorescence of the CdTe Quantum Dots-Hg(II) System

Herein, a sensitive and selective sensor for biothiols based on the recovered fluorescence of the CdTe quantum dots (QDs)-Hg(II) system is reported. Fluorescence of QDs could be quenched greatly by Hg(II). In the presence of biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), however, Hg(H) preferred to react with them to form the Hg(II)-S bond because of the strong affinity with the thiols of biothiols rather than quenching the fluorescence of the QDs. Thus, the fluorescence of CdTe QDs was recovered. The restoration ability followed the order GSH > Hcy > Cys due to the decreased steric hindrance effect. A good linear relationship was obtained from 0.6 to 20.0 mu mol L-1 for GSH and from 2.0 to 20.0 mu mol L-1 for Cys, respectively. The detection limits of GSH and Cys were 0.1 and 0.6 mu mol L-1, respectively. In addition, the method showed a high selectivity for Cys among the other 19 amino acids. Furthermore, it succeeded in detecting biothiols in the Hela cell.

A third-generation H2O2 biosensor based on horseradish peroxidase-labeled Au nanoparticles self-assembled to hollow porous polymeric nanopheres

A novel third-generation biosensor for hydrogen peroxide (H2O2) was developed by self-assembling gold nanoparticles to hollow porous thiol-functionalized poly(divinylbenzene-co-acrylic acid) (DVB-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in hollow porous thiol-functionalized poly(DVB-co-AA) nanosphere latex to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups of the nanospheres. Finally, horseradish peroxidase (HRP) was immobilized on the surface of the gold nanoparticles. The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The resulting biosensor showed a wide linear range of 1.0 mu M-8.0 mM and a detection limit of 0.5 mu M estimated at a signal-to-noise ratio of 3. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.

Surface charge influence on the surface plasmon absorbance of electroactive thiol-protected gold nanoparticles

Gold nanoparticles (3.1-5.0 nm in size) surface-derivatized with both electroactive and nonelectroactive self-assembled monolayers were synthesized. The surface-derivatized electroactive particles can be easily oxidized/reduced at an electrode surface based on the diffusion-controlled current-voltage curve observed in cyclic voltammetry measurements. Spectroelectrochemical investigation demonstrated that the maximum absorbance of the nanoparticles in their oxidized state red-shifted compared with their reduced state to a different extent according to their size distribution. In the case of the particles surface-derivatized with nonelectroactive monolayers, much less shift was observed. This study showed that surface plasmon absorbance of gold nanoparticles was not only related to core charge states but was also influenced by surface charge states as well.

Novel hole-transporting materials based on 1,4-bis(carbazolyl)benzene for organic light-emitting devices

Novel hole-transporting molecules containing 1,4-bis(carbazolyl)benzene as a central unit and different numbers of diphenylamine moieties as the peripheral groups have been synthesized and characterized. These compounds are thermally stable with high glass transition temperatures of 141-157 degreesC and exhibit chemically reversible redox processes. Their amorphous state stability and hole transport properties can be significantly improved by increasing the number of diphenylamine moieties in the outer part and by controlling the symmetry of the carbazole-based molecules. These compounds can be used as good hole-tran sporting materials for organic electroluminescent (EL) devices. The device performance based on tri- and tetra-substituted carbazole derivatives is comparable to that of a typical 4,4'-bis[N-(1-naphthyl)-N-phenylamino] biphenyl (NPB)-based device.

Immunosensors based on layer-by-layer self-assembled Au colloidal electrode for the electrochemical detection of antigen

Colloidal Au particles have been deposited on the gold electrode through layer-by-layer self-assembly using cysteamine as cross-linkers. Self-assembly of colloidal Au on the gold electrode resulted in ail easier attachment of antibody, larger electrode surface and ideal electrode behavior. The redox reactions of [Fe(CN)(6)]-/[Fe(CN)(6)](3-) on the gold surface were blocked due to antibody immobilization, which were investigated by cyclic voltammetry and impedance spectroscopy. The interaction of antigen with grafted antibody recognition layers was carried out by soaking the modified electrode into a phosphate buffer at pH 7.0 with various concentrations of antigen at 37degreesC for 30 min. Further, an amplification strategy to use biotin conjugated antibody was introduced for improving the sensitivity of impedance measurements. Thus, the sensor based oil this immobilization method exhibits a large linear dynamic range, from 5 - 400 mug/L for detection of Human IgG. The detection limit is about 0.5 mug/L.

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (Phi = 16 nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.