Effects of microcystins on the growth and the activity of superoxide dismutase and peroxidase of rape (Brassica napus L.) and rice (Oryza sativa L.)

Microcystins are naturally occurring hepatotoxic cyclic heptapeptides produced by some toxic freshwater cyanobacterial species. In this study, crude extract of toxic cyanobacterial blooms from Dianchi Lake in southwestern China was used to determine the effects of microcystins on rape (Brassica napus L.) and rice (Oryza sativa L.). Experiments were carried out on a range of doses of the extract (equivalent to 0, 0.024, 0.12, 0.6 and 3 mug MC-LR/ml). Investigations showed that exposure to microcystins inhibited the growth and development of both rice and rape seedlings, however, microcystins had more powerful inhibition effect on rape than rice in germination percentage of seeds and seedling height. Microcystins significantly inhibited the elongation of primary roots of rape and rice seedlings. Determination of the activities of peroxidase and superoxide dismutase demonstrated that microcystin stress was manifested as an oxidative stress. Using ELISA, microcystins were examined from the extract of exposed rape and rice seedlings, indicating that consumption of edible plants exposed to microcystins via irrigation route may have health risks. Significantly different levels of recovered microcystins between exposed rice and rape seedlings Suggested that there might be different tolerant mechanisms toward microcystins. (C) 2004 Elsevier Ltd. All rights reserved.

Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)(2) and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC-Cl. The new reagent BCEC-Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC-amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)(2) and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I (BCEC)/I (BCEOC) = 1.17-3.57, I (BCEC)/I (FMOC) = 1.13-8.21, and UVBCEC/UVBCEOC = 1.67-4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C-18 column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)(2). Excellent linear responses were observed, with coefficients of > 0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)(2) and Try from bee-collected pollen (bee pollen) samples.

油菜内源和外源蛋白的表达及环境因素的影响

植物与昆虫的互作关系是个长期进化的过程，虫害给农业生产带来巨大损失。本研究以甘蓝型油菜（Brassica napus）为例，研究了不同环境条件和遗传背景下外源基因的表达与效用，同时利用蛋白质组技术，研究了虫害损伤模拟条件下植物可能存在的内源抗性机制。甘蓝型油菜中转入了人工合成的Bt（Bacillus thuringiensis）杀虫基因，能使植物产生抗虫蛋白抵御虫害。我们在湖北湖南两个实验点进行了大田实验，按植株生长发育的4个不同时期从转基因植株的叶片上采样，研究抗虫蛋白在植物体内的表达动态。植株顶部第三片展开叶的Bt毒蛋白浓度在结荚期前随植物生长而不断增加，而在结荚期出现或增或减的现象。采样叶片的可溶性总蛋白浓度含量一直呈增加的趋势，直到结荚以后出现含量的明显降低。同时，收集了转基因油菜与湘油15号在田间自然杂交形成的杂交后代种子用于栽培，用GFP仪检测杂交后代的绿色荧光蛋白（green fluorescent protein），并用聚合酶链式反应（polymerase chain reaction, PCR）检测并确认带有转基因的杂交植株。为了检测带有转基因的杂交后代油菜中Bt毒蛋白的杀虫效率，用对Bt毒蛋白敏感的试虫品系——初孵棉铃虫幼虫（Helicoverpa armigera）进行杀虫活性检测实验。结果表明，携带Bt基因的杂交湘油及其转基因亲本对试虫的体重增长量均产生了负面影响，可以推断在调查取样的植株生长发育阶段，转基因杂交后代与其转基因亲本植株的杀虫效率没有显著差异。转基因植物及其杂交后代中抗虫蛋白的持续表达及田间带有转基因的自播植物的出现会使害虫产生耐受抗性的潜在可能性增加。 相对于人为增加的抗虫基因，植物在长期对抗昆虫的过程中也进化形成了自我防御机制，能够产生特异的抗性蛋白来应对昆虫的取食。本研究用机械损伤模拟害虫取食，对比了油菜受到物理损伤前后可溶性总蛋白的含量变化并试图通过蛋白质组学技术来检测可能发生变化的蛋白质。Bradford定量测定发现，同一植株同一叶片损伤前后可溶性总蛋白含量差异显著，损伤后蛋白表达量显著增高。蛋白质组双向凝胶电泳及其差异分析显示，损伤前后有8个蛋白质点发生明显的上调或下调。选择其中2个差异蛋白点经过MALDI-TOF质谱鉴定，它们分别是Rubisco小亚基前体以及果糖－1，6－二磷酸醛缩酶和粪卟啉－3－氧化酶的混合物，这些蛋白质在其他植物的抗逆研究中也有报道，它们可能在油菜叶片应答机械损伤过程中对维持植物的生理功能也有重要作用。

转基因油菜和野芥菜杂交种的适合度

转基因植物与野生亲缘种间的基因流动是目前生物安全的研究热点。甘蓝型油菜（Brassica napus 以下简称油菜）及其亲缘种是基因流研究中的模式植物体系，油菜的亲缘种之一野芥菜(B. juncea)在中国分布广泛，是常见的农田杂草。本文以转基因抗虫油菜和野芥菜为研究材料，分别用综合适合度(基于生长和生殖)和适合度(仅基于种子产量)的指标来评估其杂交种在模拟农田情况下的适合度，揭示转基因在自然环境中的命运，以期为中国的油菜基因流的管理和控制提供参考。主要结果如下： 由于杂种优势和亲本效应的影响，转基因杂交种在不同的种植季节的生长与长势较好的亲本相近。因而转基因杂交种的综合适合度介于双亲之间，或与表现较好的亲本相似。因结实较差，从种子产量上来看，转基因杂交种的适合度较低。杂交种的后代因母本效应保留了一定的休眠特性，这有助于杂交种在种子库中持续存在。野芥菜和杂交种秋播时在冬季覆大棚条件下的适合度较高，在全球变暖的情况下，野芥菜和杂交种有可能在原先较冷不适宜的地区生长，从而促进转基因的散布。 杂交实验表明，转基因花粉与野芥菜的亲和性略高于非转基因花粉，但不显著；在两种花粉发生竞争时，转基因花粉的亲和性显著较高。混合花粉授粉的结实率高于单种花粉的结实率。可见在转基因花粉在授粉阶段不存在适合度代价。同时两季秋播田间实验表明，转基因油菜与非转基因油菜的适合度没有明显差异，即在植株的生长结实阶段，当没有竞争和虫害选择压力时，转基因没有适合度代价。也证明转基因散布的潜在可能性较大。此外，转基因杂交种的开花量很大，而回交实验中转基因杂交种花粉的育性高于胚珠的育性。转基因有可能通过持续的回交逃逸。 油菜的种子较大，野菜芥的种子较小，转基因油菜与野芥菜的杂交种基本都是小种子，其种子直径低于双亲。种子大小影响了发芽率、出苗率和营养生长期。小种子出苗率和发芽率较低，营养生长期较长。在没有竞争的条件下，春播实验中，种子大小对植物的适合度及综合适合度没有影响。秋播实验中种子大小对除转基因油菜外的所有植物基因型的综合适合度有显著影响。但对于依据种子产量计算的适合度指标来说，种子大小仅对野芥菜有影响，而对其它基因型影响不大。竞争扩大了种子大小之间生长表现的差异。竞争条件下，不同种子级别间的生长变异系数大于无竞争的情况。 总之，转基因花粉较强的竞争能力和其与野芥菜的亲和性使转基因杂交种的产生成为可能。来自母本的休眠特性有助于杂种种子在种子库中存在并在条件适宜时萌发、开花，有助于转基因的进一步扩散。转基因杂交种具有较高的综合适合度，虽然结实率很低，但其开花量比较大，可能会产生大量可育的花粉，在与野生亲本的持续回交过程中，转基因有可能逃逸。同时，转基因杂交种的种子级别偏小有可能会加剧这种逃逸。

水分处理对油菜叶位光合速率、蒸腾速率及水分利用效率的影响

通过对不同水分处理下油菜不同叶位叶片光合速率(Pn)、蒸腾速率(E)和水分利用效率(WUE)的测定表明,油菜主茎叶片的光合速率、蒸腾速率和水分利用效率随叶位的降低而降低;干旱胁迫下油菜水分利用效率显著提高,光合速率变化不明显,但上部、下部叶间光合速率差异大。充分供水时叶片的蒸腾速率提高,水分利用效率则降低,从上到下叶片水分利用效率、光合速率降低不剧烈,而蒸腾速率表现为上部和下部叶高,而中部叶低。据测定,干旱处理下油菜单株生物量为37.45g,充分供水时为38.76g,干旱导致油菜生物量降低;水分胁迫能提高作物水分利用效率;在充分供水条件下叶片蒸腾速率较高,这可能是气孔行为的结果。

LC-DAD-ESI-MS Characterization of Carbohydrates Using a New Labeling Reagent

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupling to liquid chromatography with electrospray ionization mass spectrometry for the detection of carbohydrates from the derivatized rape bee pollen samples is reported. Carbohydrates are derivatized to their bis-NMP-labeled derivatives. Derivatives showed an intense protonated molecular ion at m/z [M+H](+) in positive-ion detection mode. The mass-to-charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative analysis of carbohydrates. This characteristic fragment ion is from the cleavage of C2-C3 bond in carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O](+) for pentose, hexose and glyceraldehydes and at m/z [MH-CmH2m-1Om+1-H2O](+) for alduronic acids such as galacturonic acid and glucuronic acid (m = n - 2, n is carbon number of carbohydrate). No interferences for all aliphatic and aromatic aldehydes presented in natural environmental samples were observed due to the highly specific parent mass-to-charge ratio and the characteristic fragment ions. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed-phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected.

Detection of carbohydrates using new labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone by capillary zone electrophoresis with absorbance (UV)

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupled with capillary electrophoresis (CE) with DAD detection for the determination of carbohydrates has been developed. The chromophore in the 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent is replaced by naphthyl functional group, which results in a reagent with very high molar absorptivity (epsilon(251nm) = 5.58 x 10(4) L mol(-1) cm(-1)). This pen-nits NMP-labeled carbohydrates to be detected with UV absorbance in standard 50-mu m-i.d. fused silica capillaries by zone electrophoresis. in this mode, nanomolar concentrations of detection limits are obtained. The method for the derivatization. of carbohydrates with NMP is simplified. The derivatization reaction is rapid and mild in the presence of ammonia catalyst without further transfer steps. Nine monosaccharide derivatives such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected in CE mode. Good reproducibility can be obtained with relative standard deviation (R.S.D.) values of the migration times and peak area, respectively, from 0.44 to 0.48 and from 3.2 to 4.8. Furthermore, the developed method has been successfully applied to the analysis of carbohydrates in the hydrolyzed rape bee pollen samples. (C) 2008 Published by Elsevier B.V.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.