The involvement of HSP22 from bay scallop Argopecten irradians in response to heavy metal stress

Heat shock protein 22 (HSP22) is an important member of small heat shock protein (sHSP) subfamily which plays a key role in the process of protecting cells, facilitating the folding of nascent peptides, and responding to stress. In the present study, the cDNA of HSP22 was cloned from Argopecten irradians (designated as AiHSP22) by rapid amplification cDNA end (RACE) based on the expressed sequence tags (ESTs). The full-length cDNA of AiHSP22 was of 1,112 bp, with an open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The deduced amino acid sequence of AiHSP22 showed high similarity to previously identified HSP22s. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd2+, Pb2+ or Cu2+ were investigated by real-time quantitative RT-PCR. The mRNA of AiHSP22 was constitutively expressed in all examined tissues, including haemocyte, muscle, kidney, gonad, gill and heart. The expression level in heart and muscle was higher than that in other tissues. The mRNA level of AiHSP22 in haemocytes was up-regulated after a 10 days exposure of scallops to Cu2+, Pb2+ and Cd2+. However, the expression of AiHSP22 did not increase linearly along with the rise of heavy metal concentration. Different concentrations of the same metal resulted in different effects on AiHSP22 expression. The sensitive response of AiHSP22 to Cu2+, Pb2+ and Cd2+ stress indicated that it could be developed as an indicator of exposure to heavy metals for the pollution monitoring programs in aquatic environment.

Determination of heavy metal ions by capillary electrophoresis with contactless conductivity detection after field-amplified sample injection

A method has been developed for determining of heavy metal ions by field-amplified sample injection capillary electrophoresis with contactless conductivity detection. The effects of the 2-N-morpholinoethanesulfonic acid/histidine (MES/His) concentration in the sample matrix, the injection time and organic additives on the enrichment factor were studied. The results showed that MES/His with a low concentration in the sample matrix, an increase of the injection time and the addition of acetonitrile improved the enrichment factor. Four heavy metal ions (Zn2+, Co2+, Cu2+ and Ni2+) were dissolved in deionized water, separated in a 10 mM MES/His running buffer at pH 4.9 and detected by contactless conductivity detection. The detection sensitivity was enhanced by about three orders of magnitude with respect to the non-stacking injection mode. The limits of detection were in the range from 5 nM (Zn2+) to 30 nM (Cu2+). The method has been used to determine heavy metal ions in tap water.

大蒜、绊根草中重金属抗性分子机制的研究

日益加剧的重金属污染已经危害到了全球的生态环境以及人类健康。在分子水平上阐明植物中的重金属抗性机制并应用于环境修复和绿色农业是植物科学和环境科学以及农业科学的交叉点和新的生长点。为了了解植物重金属抗性的分子机制，我们的研究主要是从重金属抗性植物材料大蒜(Allium sativumL．)和绊根草(Cynodon dactylon)中分离重金属抗性相关基因，并研究它们在重金属抗性机制中的功能。 在高等植物中有迹象表明，一种富含半胱氨酸的低分子量蛋白．类金属硫蛋白 (Metallothioneins Like，MTs Like)和一类具有Y-(Glu-Cys) n-Gly特殊结构的多肽一植物络合素(Phytochelatins，PCs)在重金属抗性机制中占有重要地位。然而人们对于同一种植物中这两种重金属结合肽作用的相互关系还缺乏了解，同时对于MT Like基因以及PCs合酶基因在同一种植物中的表达模式如金属离子专一性、时空表达特点等，还投有文献报道，因此本文将首先以这两个基因为切入点进行研究。 本研究采用RACE的方法，从大蒜中分离得到了类金属硫蛋白(MT-Like)的cDNA序列(GenBank Accession No.AY050510)，PCR和SoutheLrn Blot分析表明，大蒜基因组中不仅存在类金属硫蛋白基因，而且可能以基因家族的形式存在。对获得的MT Like cDNA进行的序列分析及同源性分析表明，大蒜MT Like cDNA含有一个完整的开放阅读框架，编码73个氨基酸，其中12个为半胱氨酸，占氨基酸总数的1 6.4%，并与其他植物如水稻、小麦、紫羊茅草中的类金属硫蛋白基因同源性较高，其中最高达89%。对该基因编码的氨基酸序列和结构分析表明在N-端、c-端结构域中分别含有3个典型的金属硫蛋白的结构模式Cys-Xaa-Cys，属于典型的Type-1类金属硫蛋白。这些Cys-Xaa-Cys特征结构表明大蒜MT Like基因编码的蛋白可以结合二价金属离子。重金属胁迫下大蒜根中MT Like基因在转录水平的表达检测表明，MT Like基因的表达受重金属离子Cu2+、Cd2+的诱导，暗示MT Like基因在大蒜对重金属的抗性中有重要作用。此外，用能谱电镜技术研究大蒜中重金属的积累与分布，以及用组织原位杂交技术分析MT Like基因的表达定位与重金属的积累、转运的关系已在进行之中。 植物络合素也是富含巯基的多肽化合物，在重金属抗性中起重要作用。由植物络合素结构中存在的Y一酰胺键或β-Ala可知PCs不是基因表达的直接产物，而是以GSH为前体的酶促反应产物。目前已知y一谷氨酰半胱氨酸二肽转肽酶(简称为PCs合酶，phytochelatin synthase，PCS)是PCs合成途径的关键酶，编码这一关键酶的基因目前已在小麦、拟南芥菜和裂殖酵母中克隆。由于这一基因在不同物种中的保守性较低，其克隆较困难。本研究通过设计植物络合素台酶基因简并引物，从大蒜中扩增得到了345bp的cDNA序列。序列分析和推测的氨基酸序列同源性比较表明，此序列的翻译产物与已知的植物络合素合酶同源性最高，此cDNA序列应为大蒜植物络合素合酶基因的部分cDNA序列(GenBank Accession No.AF384110)。目前大蒜植物络台素合酶基因的全长序列的扩增，以及这两种与重金属抗性有关的基因(MT Like，PCS)的表达模式仍在研究中。 本文还尝试了利用酵母重金属敏感突变株M379/8功能互补的方法从重金属抗性植物绊根革中分离新的重金属抗性相关基因。构建了用于转化的酵母质粒表达文库，探索了酵母转化体系建立的条件。曾尝试多种转化方法，并对其中的条件进行了优化改进。下一步的工作将集中在合适的酵母突变体的筛选或穿梭表达载体的选择标记基因替换上

绊根草重金属抗性相关基因的克隆及其功能分析及可耐受偏二甲肼的芦苇变异株系的筛选

本研究利用酵母功能互补方法和RACE的方法从具有较强抗逆能力的绊根草中克隆了9个与重金属抗性相关的克隆，并对部分基因的表达调控及功能进行了初步研究。同时还利用细胞工程技术筛选到了具有较强的耐受火箭推进齐-偏二甲肼(UDMH)的芦苇的变异株系，为以后用人工湿地系统处理受偏二甲肼污染的废水奠定了基础。 本研究通过酵母功能互补法克隆到了五个基因，分别为CdSRP、CdTETH、 CdASP、CdMT2和CdTER1。CdSRP可能是一种衰老相关基因;CdTETH编码的产物可能是组成TRAPP复合体的一个亚基;CdASP是一个功能未知的基因;CdMT2是一个编码Type Ⅱ型金属硫蛋白基因;CdTER1可能是编码一个TERl-like家族蛋白成员的基因。用这五个基因分别转化因Acr基因缺失而对As敏感的酵母菌株FD236-6A，所获得的转化子对As的抗性均有提高，其中以CdMT2、CdTER1和CdASP的作用最为明显。这些基因的表达调控方式以及与其它重金属抗性的关系正在研究中。 本研究还利用RACE的方法克隆了一个谷胱甘肽S-转移酶基因，CdGSTFl;两个植物络合素合酶基因，CdPCSI和CdPCSⅡ，和一个TypeⅠ型金属硫蛋白基因CdMT1。CdGSTF1属于phi类GST基因，Northern-blotting分析表明，CdGSTF1在绊根草根部的表达受Cd2+的诱导，暗示其可能具有解除氧自由基或氢过氧化物的毒性的作用。CdPCSI和CdPCSⅡ的同源性较高，表明绊根草含有两个以上的PCs合酶的基因。参照前人的方法对CdPCSI和CdPCSII的氨基酸序列进行分析，发现它们含有六个非常相近的Cd2+结合位点，这两个基因的功能及其调控方式有何差异尚需进一步的研究。cdMT1与用酵母功能互补法克隆到的CdMT2属于不同类型的MT基因，对它们之间很可能存在的功能、组织特异性等方面的差异性进行了讨论。 四氧化二氮／偏二甲肼是常用的航天器双组元液体推进剂。偏二甲肼易挥发，有致癌、致畸、致突变的毒性。在推进剂贮存、运输、转注、火箭发动机试车、火箭发射、管道及设备冲洗中产生的含有偏二甲肼的废水能够对卫星发射基地的地下水源和空气造成污染。因此迫切需要培育能够净化偏二甲肼污水的植物。 本研究利用生长在卫星发射基地的野生芦苇的种子诱导愈伤组织，进而通过逐步提高偏二甲肼筛选压力的方式从中筛选出具有较强抗性的愈伤组织，然后诱导其分化。目前已经得到能够在含有1.63 mmol/L和3.26 mmol/L偏二甲肼的分化培养基中生长良好的芦苇再生苗，并已成功转移至温室中。抗性分化苗对污水的处理效果和耐受偏二甲肼的机理正在研究中。

利用多基因转化技术培育重金属超富集植物的研究

植物耐受和积累重金属的细胞学基础是植物细胞内存在一些能够络合和区隔化金属离子的机制。细胞中络合重金属离子最重要的小肽分子是谷胱甘肽(GSH)和植物络合素(PCs)，而YCFⅠ基因编码的ABC-type 液泡膜转运蛋白负责将重金属离子及其与上述小肽形成的复合物转运进入细胞液泡中，即将重金属离子区隔化。植物细胞中合成GSH 和PCs 的关键酶分别是γ－谷氨酰氨半胱氨酸合成酶(GSHⅠ)和植物络合素合酶(PCS)，他们的编码基因分别为GSHⅠ 和PCS 。此外定位于细胞质中的小囊泡上且对二价阳离子的吸收和转运有重要作用的SMF2 蛋白可能也参与重金属离子的区隔化过程。 为了改良植物使之能够应用于清除土壤中的重金属污染，本研究基于植物耐受和积累重金属的细胞学机制，分别将酿酒酵母来源的GSHⅠ、YCFⅠ和SMF2 基因，以及GSHⅠ、YCFⅠ基因分别与镉抗性植物大蒜来源的AsPCSⅠ 基因构建为不同的基因组合表达载体，转化模式植物拟南芥。对不同组合转基因拟南芥的功能分析表明： 1、酵母来源的基因GHSⅠ、YCFⅠ分别在拟南芥中异源超表达可以在一定程度上提高转基因拟南芥耐受、积累重金属的能力;其中GSHⅠ基因在拟南芥超表达可以提高转基因拟南芥合成GSH 的能力，转基因拟南芥细胞中GSH 浓度比野生型增加。 2、将GSHⅠ基因和来自大蒜的AsPCSⅠ基因同时在拟南芥中超表达能够显著提高转基因拟南芥耐受和积累重金属的能力，且积累和耐受能力显著高于分别转GSHⅠ或AsPCSⅠ的单价转基因株系;将YCFⅠ基因和AsPCSⅠ基因同时在拟南芥中超表达也能够显著提高转基因拟南芥耐受和积累重金属的能力，且积累和耐受能力显著高于分别转YCFⅠ或AsPCSⅠ的单价转基因株系。两种双价转基因株系GSHⅠ＋AsPCSⅠ和YCFⅠ＋AsPCSⅠ在积累和耐受不同重金属胁迫方面没有明显差别。 3、将SMF2 基因在拟南芥中异源表达，研究了植物中囊泡转运是否参与了重金属离子的吸收和区隔化过程。研究结果表明：超表达SMF2 基因的拟南芥尽管耐受重金属胁迫的能力与野生型没有明显差异，但其积累重金属的能力显著提高。这为证明植物中小囊泡转运参与重金属转运提供了间接证据。 综上所述，同时将多个参与植物对重金属络合、转运和区隔化作用的关键基因在转基因植物中表达可以提高植物耐受和积累重金属的能力，是培育可用于植物修复的新型工程植物的值得探索的途径。本论文所设计和构建的双价基因组合及其对目标植物的转化，在环境重金属污染的清除中有潜在的应用价值。

Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

Effect of heavy metals (Cd, Cu) on the gametophytes of Laminaria japonica Aresch

Effects of various concentrations of two heavy metals, namely Cd and Cu, on gametophytes of Laminaria japonica Aresch were determined by recording morphological changes of gametophytes, determining pH values and the heavy metal content of the culture solution, calculating the germination rate of sporophytes, and observing heavy metal (Cd) distribution using a fluorescence microscope. The results showed that heavy metals damaged the gametophytes, and were even lethal, and that the higher the concentration of heavy metal ions, the greater the injury to gametophytes. Gametophytes could not survive in culture solutions containing more than 100 mg/L Cd and 50 mg/L Cu and were only able to survive in culture solution containing a mixture of Cd and Cu tip to a concentration of 10 mg/L, which indicates that gametophytes have a higher tolerance to Cd than Cu and that multiple heavy metal ions in solution markedly aggravate the damage to gametophytes compared with individual heavy metal ions. With increases in the concentration of the heavy metal, the burgeoning rate of sporophytes decreased acutely, and solutions containing multiple heavy metal ions caused even more marked harm to sporophytes than solutions containing a single heavy metal ion, because most sporophytes died in mixed solutions. The pH value of the culture medium dropped immediately at the beginning (the first day) of treatment, increased over the following days, and then decreased again. The pH of culture media containing multiple heavy metal ions showed greater variation than media containing a single heavy metal ion, with the extent of the decrease in pH of culture media containing multiple ions being greatest during the last period of the experiment. With increases in the concentration of heavy metals, the capacity of gametophytes to accumulate these ions increased. The blue fluorescent light emitted by the Cd- and Cd-binding protein complex existing in gametophytes in media containing different concentrations of Cd showed clearly the distribution of the ion in gametophytes and the results obtained were consistent with distribution determined using other methods. All results of the present study showed that gametophytes of L. japonica play a remarkable role as heavy metal decontaminators, especially with regard to Cd.

Analysis of heavy metals of muscle and intestine tissue in fish - in Banan section of Chongqing from Three Gorges Reservoir, China

The present study was carried out to investigate contamination of heavy metals in 19 fish species from the Banan section of Chongqing in the Three Gorges, Yangtze River. The results showed that the mean concentrations of heavy metals were higher in intestine than muscle, except zinc in upper strata. In the fish inhabiting the upper strata, there were significant differences between mean concentrations of As, Cr, Cu and Hg in muscle and intestine (P <0.05). There were also significant differences between mean concentrations of Cr and Cu in muscle and intestine in the fish inhabiting middle strata. However, significant differences between mean concentrations of As, Cd, Hg, Pb and Zn were measured in fish inhabiting bottom strata in both intestine and muscle tissues (P <0.05). For the fish inhabiting different strata, the concentrations of As, Cd, Cr, Cu, Hg and Ph in muscle and intestine of the fish from bottom strata (BS) were higher than those in both upper strata (US) and middle strata (MS); whereas a higher concentration of Zn was measured in muscle and intestine from fish inhabiting upper strata. Mean metal concentrations were found to be higher in age 11 than those in age I in Coreius heterodon (2- and 1-year odl fish respectively). The overall results indicated that fish muscle in the Banan section were slightly contaminated by heavy metals, but did not exceed Chinese food standards.

Mobilization potential of heavy metals: A comparison between river and lake sediments

Toxic metals introduced into aquatic environments by human activities accumulation in sediments. A common notion is that the association of metals with acid volatile sulfides (AVS) affords a mechanism for partitioning metals from water to solid phase, thereby reducing biological availability. However, variation in environmental conditions can mobilize the sediment-bound metal and result in adverse environmental impacts. The AVS levels and the effect of AVS on the fate of Cu, Cd, Zn, Ni in sediments in the the Changjiang River, a suboxic river with sandy bottom sediment and the Donghu Lake, a anoxic lake with muddy sediment in China, were compared through aeration, static adsorption and release experiments in laboratory. Sips isotherm equation, kinetic equation and grade ion exchange theory were used to describe the heavy metal adsorb and release process. The results showed that AVS level in the lake sediment are higher than that of the river. Heavy metals in the overlying water can transfer to sediments incessantly as long as the sediment remains undisturbed. The metal release process is mainly related to AVS oxidation in lake sediment while also related to Org-C and Fe-Mn oxyhydroxide oxidation in river sediment. The effect of sulfides on Zn and Ni is high, followed by Cd, and Cu is easy bound to Org-C. AVS plays a major role in controlling metals activity in lake sediment and its presence increase the adsorption capacity both of the lake and river sediments.

Heavy metals in plants and phytoremediation - A state-of-the-art report with special reference to literature published in Chinese journals

Goal, Scope and Background. In some cases, soil, water and food are heavily polluted by heavy metals in China. To use plants to remediate heavy metal pollution would be an effective technique in pollution control. The accumulation of heavy metals in plants and the role of plants in removing pollutants should be understood in order to implement phytoremediation, which makes use of plants to extract, transfer and stabilize heavy metals from soil and water. Methods. The information has been compiled from Chinese publications stemming mostly from the last decade, to show the research results on heavy metals in plants and the role of plants in controlling heavy metal pollution, and to provide a general outlook of phytoremediation in China. Related references from scientific journals and university journals are searched and summarized in sections concerning the accumulation of heavy metals in plants, plants for heavy metal purification and phytoremediation techniques. Results and Discussion. Plants can take up heavy metals by their roots, or even via their stems and leaves, and accumulate them in their organs. Plants take up elements selectively. Accumulation and distribution of heavy metals in the plant depends on the plant species, element species, chemical and bioavailiability, redox, pH, cation exchange capacity, dissolved oxygen, temperature and secretion of roots. Plants are employed in the decontamination of heavy metals from polluted water and have demonstrated high performances in treating mineral tailing water and industrial effluents. The purification capacity of heavy metals by plants are affected by several factors, such as the concentration of the heavy metals, species of elements, plant species, exposure duration, temperature and pH. Conclusions. Phytoremediation, which makes use of vegetation to remove, detoxify, or stabilize persistent pollutants, is a green and environmentally-friendly tool for cleaning polluted soil and water. The advantage of high biomass productive and easy disposal makes plants most useful to remediate heavy metals on site. Recommendations and Outlook. Based on knowledge of the heavy metal accumulation in plants, it is possible to select those species of crops and pasturage herbs, which accumulate fewer heavy metals, for food cultivation and fodder for animals; and to select those hyperaccumulation species for extracting heavy metals from soil and water. Studies on the mechanisms and application of hyperaccumulation are necessary in China for developing phytoremediation.

Effects of heavy metals on plants and resistance mechanisms

Goal, Scope and Background. As one of the consequences of heavy metal pollution in soil, water and air, plants are contaminated by heavy metals in some parts of China. To understand the effects of heavy metals upon plants and the resistance mechanisms, would make it possible to use plants for cleaning and remediating heavy metal-polluted sites. Methods. The research results on the effects of heavy metals on plants and resistant mechanisms are compiled from Chinese publications from scientific journals and university journals, mostly published during the last decade. Results and Discussion. Effects of heavy metals on plants result in growth inhibition, structure damage, a decline of physiological and biochemical activities as well as of the function of plants. The effects and bioavailability of heavy metals depend on many factors, such as environmental conditions, pH, species of element, organic substances of the media and fertilization, plant species. But, there are also studies on plant resistance mechanisms to protect plants against the toxic effects of heavy metals such as combining heavy metals by proteins and expressing of detoxifying enzyme and nucleic acid, these mechanisms are integrated to protect the plants against injury by heavy metals. Conclusions. There are two aspects on the interaction of plants and heavy metals. On one hand, heavy metals show negative effects on plants. On the other hand, plants have their own resistance mechanisms against toxic effects and for detoxifying heavy metal pollution. Recommendations and Outlook. To study the effects of heavy metals on plants and mechanisms of resistance, one must select crop cultivars and/or plants for removing heavy metals from soil and water. More highly resistant plants can be selected especially for a remediation of the pollution site. The molecular mechanisms of resistance of plants to heavy metals should be studied further to develop the actual resistance of these plants to heavy metals. Understanding the bioavailability of heavy metals is advantageous for plant cultivation and phytoremediation. Decrease in the bioavailability to farmlands would reduce the accumulation of heavy metals in food. Alternatively, one could increase the bioavailability of plants to extract more heavy metals.

Hg胁迫对两种基因型小麦生长及其过氧化物酶活性的影响

用不同浓度Hg处理两种基因型小麦种子,较低浓度的Hg对小麦种子萌发影响比较小,对抗旱品种的小麦种子(陕合)的萌发有略微的刺激作用。小剂量、短时间的重金属处理可以提高POD的活性,发芽后受到Hg胁迫的陕合对Hg的耐受性低于发芽前就受到Hg胁迫的陕合,也低于同样胁迫处理的小麦品种(郑引)。发芽后进行Hg胁迫处理情况下,陕合对于Hg胁迫比较敏感,POD活性随着Hg浓度的升高而下降;而郑引,低浓度Hg对其POD活性有促进作用。在小麦发芽前就受到Hg胁迫的情况下,陕合和郑引的POD活性都随着Hg浓度增加表现为先上升而后下降趋势。