植物络合素合酶基因的功能分析及其应用

植物络合素（phytochelatins，PCs）是含有γ-Glu-Cys重复结构的小分子多肽，其结构通式为：（γ-Glu-Cys）n-Gly（n=2-11）。植物络合素（PCs）由植物络合素合酶（PCS）催化谷胱甘肽（GSH）聚合而成，能够络合重金属离子而具有解毒功能，这是植物解毒重金属胁迫的重要机制之一。本文克隆了来源于重金属抗性植物绊根草（Cynodon dactylon cv Goldensun）的植物络合素合酶基因，通过基因工程手段使其在烟草中过量表达，得到了一些有望用于植物修复（phytoremediation）的工程植株。同时，在水稻（Oryza sativa）种子中利用RNAi技术抑制植物络合素合酶基因的表达，以降低重金属离子在人类最重要的粮食作物水稻的籽粒中的积累。 1. 通过RACE（Rapid Amplification of cDNA Ends）方法从抗性植物绊根草中克隆了植物络合素合酶基因CdPCS1，其1515 bp的读码框编码一个含505个氨基酸的蛋白质，蛋白质序列分析表明它具有植物络合素合酶的结构特征，同时还具有磷酸化位点和亮氨酸拉链结构。 2. CdPCS1基因可以互补对铜和镉离子敏感的酵母突变株ABDE-1（cup1Δ）中缺失的金属硫蛋白基因CUP1的功能，也可以互补对砷离子敏感的酵母突变体FD236-6A（acr-3Δ）中的离子外排载体基因ARC3的缺失。 3. 将CdPCS1转入烟草，共获得过表达CdPCS1的烟草44个株系，其中融合GFP的株系16个。对T0代的转基因植株的PCs含量以及重金属抗性和吸收能力进行了分析，其中抗性实验表明，在300μmol/L 的Cd2+离子胁迫11天之后，野生型植株的叶片出现斑点状坏死，而两个转基因烟草株系S6和K49的植株没有出现受伤害症状。在100μmol/L的CdSO4处理一周后，转基因植株中的PCs含量比对照有不同程度的提高，最多提高了2.88倍。当用300μmol/L Cd2+处理9天再用600μmol/L Cd2+处理2天后，Cd的积累量比野生型植株增加了2倍多;用50μmol/L As3+处理7天再用100μmol/L As3+处理2天后，转基因植株对As的积累量最多增加了3倍多。说明转入绊根草PC合酶基因的烟草增加了植物络合素的合成，并由此增加了对镉离子的抗性以及对镉离子和砷离子的积累。 4. 对转基因烟草中的CdPCS1进行了亚细胞定位研究。在激光共聚焦显微镜和荧光显微镜下分别用转基因烟草叶片组织和叶肉细胞原生质体观察融合GFP的CdPCS1，结果表明融合蛋白定位于细胞核中。 5. .利用RNAi技术抑制水稻种子中植物络合素合酶基因的表达，共获得39个转基因株系。其中35个株系为种子特异性ZMM1启动子驱动OsPCS1基因的RNAi，其余4个株系由组成型的Ubiquitin启动子驱动。RT-PCR的分析结果表明：一个由ZMM1启动子驱动的RNAi转基因水稻株系的种子中，OsPCS1的mRNA水平比对照中的下降了一半。

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。

Toxicity of heavy metals to brine shrimp Artemia

Bioassay were carried out on 48h cultured nauplii of brine shrimp Artemia by exposing them to seven trace elements viz. copper (Cu), zinc (Zn), cadmium (Cd), nickel (Ni), lead (Pb), iron (Fe) and manganese (Mn). Synergistic effects of all these elements and additive effects of Cu and Zn, Cd and Pb, and Ni and Fe were also investigated. Comparatively, the degree of toxicity for compound bioassays was higher than individual simple tests. The values were averaged and the expected median lethal concentration [LC sub(50)] of tested heavy metals was obtained by probit analysis on the basis of cumulative numbers of dead organisms after 24 and 48h. The order of toxicity of the metals to Artemia was Pb>Cd>Cu>Ni>Zn>Fe>Mn. Potency ratios of the seven metals were also calculated. The 24 and 48h variations obtained in LC sub(50) values were significantly different and relative implications of these are discussed.

化学修饰铋膜电极检测重金属镉

随着我国工业的快速发展，环境污染日益严重，其中重金属已经成为最主要的污染物 之一。重金属具有分布广泛、半衰期长等特点，因而对人们的生产生活危害巨大。镉作为 一种常见的重金属污染物，它能够引发废用性萎缩、肾功能衰竭和感染等疾病，因此对环 境中存在的痕量镉的进行检测显得极为重要。传统的痕量分析方法包括光谱分析法和色谱 分析法，但这两方法所使用的仪器比较笨重，操作过程复杂，因而不适于在线分析。电化 学分析方法因其快速、便携、价格低廉、灵敏度高等特点而受到了人们的广泛关注，其中 较为常用的阳极溶出伏安法已经在镉离子等重金属离子的现场快速高灵敏检测中发挥了 重要作用。然而传统阳极溶出法中使用的汞电极因具有毒性而被许多国家禁止使用，所以 寻找汞电极的替代电极成为近年来的阳极溶出技术的研究热点。铋膜电极因具有类似汞电 极的分析性能且环境友好而受到了广泛重视，特别是各种化学修饰方法使得铋膜电极的性 能得到了显著提高。但是目前铋膜电极仍存在稳定性低、抗干扰能力差等问题，这些不足 严重制约了该类电极在重金属的阳极溶出分析中的应用。本文旨在通过新的化学修饰方法 解决铋膜电极应用中的瓶颈问题，发展具有优异分析性能的化学修饰铋膜电极应用于镉离 子等重金属离子的阳极溶出分析。本文的主要研究内容包括： l）以阳极溶出法测定镉离子为例，研究了化学修饰铋膜电极的响应特性，考察了富 集时间、富集电位、铋离子浓度、离子载体浓度和Nafion 浓度等实验条件对检测灵敏度的 影响。 2）将离子载体引入铋膜电极与Nafion 结合使用，研究了镉离子在该电极上的阳极溶 出响应，并探讨了铜、铅、铟三种金属离子对镉离子检测选择性的影响。将这种改良后的 化学修饰铋膜电极用于实际海水样品的检测，所得结果与ICP-MS 的测量结果基本一致。 3）将四氟硼酸钠引入铋膜电极与离子载体、碳纳米管结合使用，研究了镉离子在该 电极上的阳极溶出响应，考察了铜、铅、铟离子对镉离子测定的影响。 4）考察了电解富集和开路电位富集两种富集方式对电极灵敏度和选择性的影响。 实验表明：通过预富集，在未除氧的溶液中即可得到显著的镉离子溶出电流峰，且背 景噪音低；加入离子载体后，电极对目标金属有良好的选择性，可以在复杂基体条件下测 定重金属离子镉；电解富集条件下电极的的灵敏度较高，而开路电位富集条件下电极的选 择性较好。这种环保的无汞化学修饰电极为海水中重金属污染物的检测提供了新的手段。

Hydrographical conditions and benthic assemblages in the Suez Gulf, Egypt

The coastal development and human activities along the Suez Gulf leading to sedimentation, degrade the quality of water, disturbing the natural structure and functions of aquatic communities. The Suez Gulf is a large semi-closed area with a 346 km long coastline on the western beach side. The prevailing physicochemical parameters in shallow intertidal waters were measured seasonally over the year. Benthic faunas in the sampling sites were studied indicating their regional distribution in relation to the impact of different environmental parameters in the intertidal region. The concentration of copper in seawater reached high level at St. IV (4.57 ug/1), which is exposed to sewage and petroleum hydrocarbons. The grain size of the sediment is a determining factor for the organic carbon concentration and the sandy substrate enhances organic matter degradation processes. A large number of oil fields are present along the western coast of the Suez Gulf, therefore, cadmium and organic matter appeared to be high. The values of pH did not vary greatly among the different sampling sites. It was high at EI-Ein, El-Sukhna and Ras-Shukeir due to the disposal of mainly acidic sewage and industrial effluents of the two stations Adabiya and Ras-Gharib respectively. The macrobenthos included 71 species embraced mainly from Mollusca (53.5% Gastropoda and 12.7% Bivalvia) and the other invertebrates included 7 groups namely, Rhizostoma, Polychaeta, Cirripedia, Amphipoda, Isopoda, Decapoda and Echinodermata. The distribution of benthos is affected by the temperature and salinity of seawater. The concentration of organic matter in seawater and in sediments in shallow waters shows high values in the central part of the Gulf of Suez.

Distribution of heavy metals in the northern shrimp Pandalus borealis from the Oslofjord

Studies on the distribution of heavy metals like copper, cadmium, zinc, lead and mercury in deep sea prawns Pandalus borealis in the Oslofjord region showed that those collected from inner and middle fjord contained higher levels of heavy metals than those from the outer fjord. Their content in the edible portions, viz., tail muscle, was less compared to other organs. In terms of metal concentration copper and zinc are present in significant quantities in Pandalus borealis.

Helminth parasites of Caspian kilka (genus Clupeonella) as bioindicators of heavy metals

In the present study, intestinal helminth parasite fauna of 398 specimens of three species of kilkas, C. engrauliformis (N= 92), C. grimmi (N= 136) and C. cultriventris (N= 170) from Babolsar harbor were investigated. Five parasite species were found including: Corynosoma strumosum (Acanthocephala), Pronoprymna ventricosa (Trematoda), Contracaecum sp. (Nematoda), Raphidascaris sp. (Nematoda) and Anisakis sp. (Nematoda). The highest prevalence and abundance were observed in C. strumosum and P. ventricosa. The prevalence and abundance of C. strumosum in C. grimmi was significantly higher than C. engrauliformis. The prevalence and abundance of P. ventricosa in C. cultriventris was significantly higher than C. engrauliformis. The prevalence, mean intensity and mean abundance of the parasites were compared according to the sex, length group and season also effect of parasite on host growth parameters was considered. The diversity, equability, similarity, species richness, dominance Indices and specificity indices of helminth parasites of the three host species were studied. Also the lead and cadmium concentrations in the intestine, muscle, liver, kidney and gonad of kilkas and their parasites C. strumosum and P. ventricosa were measured and compared. The results revealed that lead and cadmium concentrations in C. strumosum and P. ventricosa were significantly higher than kidney, liver, intestine, gonad and muscle. The lead and cadmium concentrations of the parasites were compared according to the sex, parasitism and season.

Trace metal (Cu, Zn, Pb, Cr and Cd) concentrations of three subfractions of organic matter from Lake Mariut sediments, Egypt

Trace elements associated with organic subfractions (humic, fulvic, and non-humic substances) were identified for seven core sediments from Lake Mariut, Egypt. Results indicated that the amounts of trace metals in humic acid and non-humic substances decreased in the following order: Zn>Cu>Pb>Cr>Cd, while in fulvic acid the order the order was Cu>Zn>Pb>Cr>Cd. There is a higher contribution of Zn, Pb, Cu and Cr in humic acid compared to fulvic acid in most samples. Slight changes in the amounts of cadmium bounded with humic and fulvic acids was also found.

Speciation of some trace metals (Fe, Cu, and Cd) in El-Mex Bay waters, Alexandria, Egypt

Surface- and bottom-water samples were collected from October 1996 to August 1997 to study levels of iron, copper, and cadmium species in their dissolved labile as well as non-labile and particulate forms in the waters of El-Mex Bay. The results showed that the non-labile concentration of the metals was generally more abundant than that of the labile form: its content reached more than 90% of the total dissolved metal for Cu and more than 80% for Fe. The particulate form was almost at the level of the labile form. The annual concentration of the trace metals of the labile form was 13 µglˉ¹ for Fe; 3µglˉ¹ for Cu, and 1.2 µglˉ¹ for Cd in the surface- and bottom-waters.

Investigation on the ecological impact of constructing and activities of petrochemical industries of petrochemical special economic zone of Mahshahr on the abundance and diversity of macrobenthic fauna in Jafari Creek, north-west of the Persian Gulf

The impact of Petrochemical Special Economic Zone (PETZONE) activities on the health status of Jafari Creek was studied by assessing the changes in macroinvertebrate assemblages in nine sites during September 2006- January 2008. Furthermore to evaluate the ecological status of the Jafari Creek the WFD indices (i.e. AMBI, M-AMBI) were used. The relationship between spatial pattern of macro invertebrate assemblages and ambient factors (i.e. water temperature, salinity, pH, dissolved oxygen, turbidity, electrical conductivity, total dissolved solid, total hardness, total nitrogen, ammonia, total phosphorous, chemical oxygen demand, biological oxygen demand, sediment grain size distribution, sediment organic content, heavy metals contents) was measured. Background Enrichment indices, Contamination factor and Contamination degree, were used to assess the health status in the study area based on Nickel, Lead, Cadmium and Mercury contents of the sediments. The macrobenthic communities had a low diversity and were dominated by opportunistic taxa, and the AMBI and M-AMBI indices need to be calibrated before using in Persian Gulf and its coastal waters. The BIO-ENV analysis identified pH, dissolved oxygen, TDS, and the total organic content of sediments as the major environmental variables influencing the infaunal pattern. This suggests that management should attempt to ensure minimal disturbance to environmental variables underlying the spatial variation in macroinvertebrate assemblages. Background Enrichment indices showed that the health of Jafari Creek has declined over time due to the constant discharge of heavy metals to the Creek system. Furthermore WQS index shows that the quality condition of the water column in Jafari Creek, regard to the calculated number (3) is week. These indices also identified a significant degree of pollution in the study area. The decrease in the ecological potential of Jafari Creek was best highlighted by the alteration in macrobenthic assemblages.

The distribution and concentration levels of trace metals in water and sediments of Lake Victoria, Kenya

The water and bottom sediments of Lake Victoria (Kenya) were analysed for A1, Fe, Mn, Zn, Pb, Cu, Cr and Cd. The total metal concentrations were determined and their mean variations and distributions discussed. The bottom lake waters showed higher concentration levels than the surface waters. The range of values (in mg/l) in the bottom and surface lake waters were as follows: Surface Waters: A1(0.08 - 3.98), Fe(0.09 - 4.01), Mn(0.02 - 0.10). Zn(0.01 -0.07), Pb(0.001- 0.007), Cu(not detected - 0.006), Cr(not detected - 0.004). Bottom Waters: A1(0.1 0 - 6.59), Fe(0.23 - 9.64), Mn(0.04 - 0.39), Zn(0.01- 0.08), Pb(0.002 - 0.009), Cu(not detected - 0.03). Cr(not detected -0.002). River mouths and shallow areas in the lake showed higher total metal concentrations than offshore deeper areas. Apart from natural metal levels, varied urban activities and wastes greatly contribute to the lake metal pollution as shown by high Pb and Zn levels in sediments, around Kisumu and Homa Bay areas. Other comparatively high values and variations could be attributed to the varied geological characteristics of the lake and its sediments. Compared to the established W.H.O (1984) drinking water standards manganese, aluminium and iron levels were above these limits whereas zinc, lead, chromium, copper and cadmium were below.

Removal of surface states and recovery of band-edge emission in InAs nanowires through surface passivation.

Surface states in semiconductor nanowires (NWs) are detrimental to the NW optical and electronic properties and to their light emission-based applications, due to the large surface-to-volume ratio of NWs and the congregation of defects states near surfaces. In this paper, we demonstrated an effective approach to eliminate surface states in InAs NWs of zinc-blende (ZB) and wurtzite (WZ) structures and a dramatic recovery of band edge emission through surface passivation with organic sulfide octadecylthiol (ODT). Microphotoluminescence (PL) measurements were carried out before and after passivation to study the dominant recombination mechanisms and surface state densities of the NWs. For WZ-NWs, we show that the passivation removed the surface states and recovered the band-edge emission, leading to a factor of ∼19 reduction of PL linewidth. For ZB-NWs, the deep surface states were removed and the PL peaks width became as narrow as ∼250 nm with some remaining emission of near band-edge surface states. The passivated NWs showed excellent stability in atmosphere, water, and heat environments. In particular, no observable changes occurred in the PL features from the passivated NWs exposed in air for more than five months.

A phototaxis inhibition assay using barnacle larvae

The effects of sublethal concentrations of phenol and cadmium on the phototactic responses of the stage II nauplii of the barnacle Balanus amphitrite were investigated. Increased toxicant concentrations caused a reduction in phototactic responses. Balanus amphitrite nauplii exposed to nominal phenol concentrations of 100 ppm and higher for 1-12 h failed to exhibit phototactic responses, while longer exposure times of 24 and 48 h reduced the lowest observable effect concentration (LOECs) to 80 and 60 ppm, respectively. For cadmium, the LOECs, based on nominal concentrations, for B. amphitrite following 1, 6, 12, 24, and 48 h exposures were 20, 4.5, 4.0, 1, and 0.75 ppm, respectively. The LOECs can be significantly reduced by increasing the duration of exposure to the toxicants. A good relationship exists between the phototactic response and toxicant concentration as well as exposure time. Results of this study indicate that the toxicant-induced reduction in phototactic responses of barnacle larvae can be used in a sensitive, rapid screening test for ecotoxicological assessments. (C) 1997 by John Wiley & Sons, Inc.

Growth and Characterization of GaSb-Based Type-II InAs/GaSb Superlattice Photodiodes for Mid-Infrared Detection

InAs/GaSb superlattice (SL) midwave infrared photovoltaic detectors are grown by molecular beam epitaxy on GaSb(001) residual p-type substrates. A thick GaSb layer is grown under the optimized growth condition as a buffer layer. The detectors containing a 320-period 8ML/8ML InAs/GaSb SL active layer are fabricated with a series pixel area using anode sulfide passivation. Corresponding to 50% cutoff wavelengths of 5.0 mu m at 77 K, the peak directivity of the detectors is 1.6 x 10(10) cm.Hz(1/2) W-1 at 77 K.

Revised ab initio natural band offsets of all group IV, II-VI, and III-V semiconductors

Using an all-electron band structure approach, we have systematically calculated the natural band offsets between all group IV, III-V, and II-VI semiconductor compounds, taking into account the deformation potential of the core states. This revised approach removes assumptions regarding the reference level volume deformation and offers a more reliable prediction of the "natural" unstrained offsets. Comparison is made to experimental work, where a noticeable improvement is found compared to previous methodologies.