高耐镉膨胀肾形虫种群金属硫蛋白基因的鉴定与诱导表达

为研究金属硫蛋白(Metallothionein, MT)对生物体抵抗重金属毒性的效应,获得一种具有镉高耐受性的膨胀肾形虫(Colpoda inflata)东北种群,该种群96h最高镉耐受浓度10 mg/L,其金属硫蛋白含量表现出与镉浓度、肾形虫种群增长率存在正相关关系。克隆获得金属硫蛋白Col-MT1基因,对基因序列和氨基酸序列特征分析表明,其为金属硫蛋白基因家族7a亚型的新成员。qRT-PCR实验证实, Col-MT1基因在60h、84h和108h三个时间点对5种浓度镉胁迫均上调表达,与镉浓度之间呈现出一定的剂量-效应关系。其分子调控机制还有待进一步研究。上述结果补充了原生动物MT基因数据库,为进一步揭示C. inflata MT基因的功能,以及应用于镉污染监测和环境修复奠定了基础。     

金属硫蛋白及其生物学功能

金属硫蛋白(metallothionein, MT) 是一类富含半胱氨酸的低分子质量蛋白质,已鉴定4种亚型：MT-1、MT-2、MT-3和MT-4,基于各亚型功能的相对异质性而使MT呈现其生物学作用的多样性。金属硫蛋白通过与金属离子结合而参与基因表达调控和机体的重金属解毒过程;金属硫蛋白通过抑制多种氧化应激途径而保护细胞免受损伤;金属硫蛋白通过参与细胞的增殖、分化和凋亡的调节而影响肿瘤及其他重大疾病的发生发展。本文在金属硫蛋白的结构和分类的基础上综述其生物学作用及其相关机制。     

锌诱导金属硫蛋白在文蛤不同组织中的表达

采用体外暴露法对文蛤Meretrixmeretrix进行锌(0、1.5mg/L、3mg/L、6mg/L)染毒,研究不同染毒浓度的锌离子在不同染毒时间下,诱导金属硫蛋白(metallothionein,MT)在文蛤足、肝胰腺、鳃、外套膜组织中的表达差异。取经不同浓度的锌溶液在染毒2d及4d后的不同组织匀浆、离心后经Bio-GelP-10凝胶柱层析纯化,用TU-1810紫外可见分光光度计测定每个样品中金属硫蛋白的吸光值。实验结果显示:同一浓度锌离子染毒后,MT在文蛤4个不同组织中的表达量差异较显著,一般为足>肝胰腺>外套膜>鳃;不同染毒浓度的锌离子在不同染毒时间下诱导MT在不同组织中的表达也不同,具有一定的组织差异性和规律性。     

镉诱导金属硫蛋白在华溪蟹组织中的表达

采用体外暴露染毒法,研究了不同浓度与时间条件下,镉诱导河南华溪蟹(Sinopotamon henanense)金属硫蛋白(metallothionein,MT)在肝胰腺、肌肉、鳃和卵巢中的表达差异。镉浓度分别为0、14·5mg/L、29mg/L和58mg/L;处理时间依次为1d、3d和5d。利用镉血红蛋白饱和法和火焰原子吸收分光光度法(AAS)测定MT的蛋白含量。结果显示,用不同的染毒浓度和处理时间,镉在组织中诱导产生MT的含量有较大差异,其中肝胰腺MT的诱导量最大,变化规律也最明显;肌肉中也有较大量MT的表达;而鳃和卵巢MT的诱导量均较低。此外,本文分析了镉的浓度与时间梯度对诱导MT表达的影响与毒性效应机制。结论:组织不同,染毒浓度及时间不同,镉诱导MT的表达也不同,具有一定的组织差异性和规律性。     

拟南芥MT-II过量表达提高抗旱性(英文)

富含巯基的植物II型金属硫蛋白(MT)对植物抵抗重金属胁迫具有重要作用,其中一个可能机制是金属硫蛋白可能猝灭重金属引起的氧化胁迫。利用转MT-II基因和野生型拟南芥(Arabidopsis thaliana)植株来对比研究MT在胁迫过程中通过清除氧自由基,特别是H2O2而对植物抗旱性的影响。研究表明,转基因型拟南芥能有效维持体内氧化—还原势,减少MDA的产生,从而缓解干旱胁迫引起的伤害,提高抗旱性。     

拟南芥MT-Ⅱ过量表达提高抗旱性

富含巯基的植物Ⅱ型金属硫蛋白(MT)对植物抵抗重金属胁迫具有重要作用,其中一个可能机制是金属硫蛋白可能猝灭重金属引起的氧化胁迫.利用转MT-Ⅱ基因和野生型拟南芥(Arabidopsis thaliana)植株来对比研究MT在胁迫过程中通过清除氧自由基,特别是H2O2而对植物抗旱性的影响.研究表明,转基因型拟南芥能有效维持体内氧化-还原势,减少MDA的产生,从而缓解干旱胁迫引起的伤害,提高抗旱性.     

植物重金属结合蛋白（肽）

本文综述了植物体中重金属结合蛋白(肽)——类金属硫蛋白(类MT)和植物螯合肽(PC)的诱导产生、物理和生物化学特性及它们的生理功能,并与动物MT作了比较。     

金属硫蛋白的结构模型

金属硫蛋白是一种低分子量(6,000—10,000),富含半胱氨酸(25—35％),并能结合重金属如Zn、Cd、Cu、Hg或Ag的蛋白质。因其含硫及金属故称为金属硫蛋白(Metallothionein,简称MT)。自1957年Margashes和Vallee在马的肾脏皮质中第一次分离出含有镉的蛋白质以来,在哺乳动物(包括人)、微生物、原核生物蓝绿藻及植物中已先后分离出金属硫蛋白。它是一种诱导性的蛋白质。在实验的动物和培养的细胞中(如Hela细胞、人的上皮细胞等)金属硫蛋白不仅能被重金属离子而且也能为皮质激素诱导而合成。并已证明二者诱导MT的合成是受转录水平调节。     

金属硫蛋白的结构模型

金属硫蛋白是一种低分子量(6,000—10,000),富含半胱氨酸(25—35％),并能结合重金属如Zn、Cd、Cu、Hg 或Ag的蛋白质。因其含硫及金属故称为金属硫蛋白(Metallothionein,简称MT)。自1957年Margashes和Vallee在马的肾脏皮质中第一次分离出含有镉的蛋白质以来,在哺乳动物(包括人)、微生物、原核生物蓝绿藻及植物中已先后分离出金属硫蛋白。它是一种诱导性的蛋白质。在实验的动物和培养的细胞中(如 Hela 细胞、人的上皮细胞等)金属硫蛋白不仅能被重金属离子而且也     

金属硫蛋白拮抗同型半胱氨酸对血管内皮细胞的脂质过氧化作用

目的:观察金属硫蛋白(metallothionein,MT)对同型半胱氨酸(homocysteine Hcy)损伤血管内皮细胞的拮抗作用及机制.方法:培养液中分别加入Hcy及Hcy MT孵育血管内皮细胞,自动生化分析仪测培养液乳酸脱氢酶(LDH)活性,硫代巴比妥酸法测细胞丙二醛(MDA)含量.结果:同型半胱氨酸增加内皮细胞LDH及蛋白漏出,并使细胞MDA含量明显升高.MT呈浓度依赖性地抑制Hcy所致的血管内皮细胞损伤及MDA增高.结论:MT拮抗Hcy对血管内皮细胞的脂质过氧化损伤.     

镉诱导植物的自由基过氧化损伤

紫羊茅(Festuca rubra L.)有对重金属敏感(“S59”)和忍耐(“Merlin”)两个品种。以0～2.5mmol/LCd~(2 )的营养液分别培养“S59”和“Merlin”幼苗,随着Cd~(2 )浓度增加和处理时间延长,幼苗生长量相应下降。0.25mml/L Cd~(2 )对“S59”的生长抑制与0.5mmol/L Cd~(2 )对“Merlin”的抑制相似,表明“Merlin”比“S59”更能耐受Cd~(2 )的毒害。在Cd~(2 )抑制生长的同时,幼苗的MDA(丙二醛,膜脂过氧化产物)和POD(过氧化物酶)活性急剧上升,CAT(过氧化氢酶)和SOD活性降低,蛋白质含量明显下降。MDA大幅度上升是Cd~(2 )培养幼苗突出的生理变化之一,2.5mmol/L Cd~(2 )培养幼苗6d,“Merlin”的MDA为对照的209％,而“S59”为对照的243％。MDA的积累与培养幼苗的Cd~(2 )浓度成密切正相关,与幼苗的生长量呈负相关。当SOD活性平稳或略为上升时,MDA稳定;SOD活性开始下降,MDA便急剧上升。很可能自由基和MDA在Cd~(2 )毒害植物中充当了重要的角色。     

Effects of Zinc on Meristem Size and Proximity of Root Hairs and Xylem Elements to the Root Tip in a Zinc-tolerant and a Non-tolerant Cultivar of Festuca rubra L.

A 4 d exposure to zinc (0.1 and 02 µg Zn cm^–3) reducedthe length of the root apical meristem in a Zn-sensitive cultivar(S59) of Festuca rubra L. to a much greater extent than in aZn-tolerant cultivar (Merlin). In S59, Zn treatment also inducedroot hair and xylem formation much closer to the root cap boundarythan in control roots, whereas Merlin was only marginally affectedby Zn treatment. The data are discussed in relation to previouslyestablished effects of Zn on the cell cycle and other cellularcharacters of the two cultivars. zinc, meristem size, root hair, xylem, Festuca rubra     

Zinc-induced vacuolation in root meristematic cells of Festuca rubra L.

Abstract. The effect of Zn on vacuole development in root meristematic cells was examined in three cultivars of Festuca rubra: a Zn-tolerant cultivar (Merlin), a salt-tolerant cultivar (Hawk) and a non-tolerant cultivar (S.59) in order to determine whether or not vacuole development was related to Zn tolerance. Treatment with Zn greatly increased the percentage of cells in the apical meristem which were vacuolated in all three cultivars (7.01-fold increase in Merlin, 3.61-fold increase in Hawk and 5.39-fold increase in S.59 over the range 0–0.5 μg Zn cm^?3). Morphometric analyses on electromicrographs indicated that the percentage total vacuolar volume fraction of meristematic tissue was also increased by Zn treatment. Most of this increase was due to an increase in a particular component of the vacuole which was 0.2–0.5 μm in diameter, spherical or ovoid in outline and possessed a distinct amorphous electron dense matrix (Type A intravacuolar body). X-ray microanalysis revealed that this matrix was rich in calcium and phosphorus in control plants. In Zn-treated roots, elevated levels of Zn were found in the matrix in Merlin and Hawk, but not in S.59. In addition, intravacuolar membranous whorls or myelin bodies were more highly organized in Zn-treated Merlin and Hawk but not S.59. Elevated Zn levels were not found in any other vacuolar component nor in nuclei, nucleoli, cell walls or ground cytoplasm. Control roots of Merlin possessed a greater type A vacuolar volume fraction than either Hawk or S.59 which may confer a greater capacity to compartmentalize Zn at the onset of exposure to toxic metals than the other cultivars. Predictably, the EM procedures resulted in loss of Zn from the roots to the fixative and dehydrating solutions. However, the rate of loss was the same (approximately 75–80%) regardless of cultivar. These findings are discussed in relation to their possible role in the mechanisms of Zn tolerance in these cultivars.     

Effect of amendments on phytoavailability and fractionation of copper and zinc in a contaminated soil

The ability of amendments to modify the soil properties and influence plants to immobilise Cu and Zn was studied in a naturally contaminated, additionally spiked podzolic soil. Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass) were tested in a pot study in the presence of soil amendments (lime, phosphate, and compost, individually and in combination) to assess the effect of soil-plant-amendment interaction on phytostabilisation. The ability of treatments to stabilize metals was assessed on the basis of metal fractionation in soil, partitioning of metals in plants, and metal uptake by the plants. Significant partitioning of Cu into immobile forms occurred as a result of the growth of Festuca rubra, and of Zn by the growth of Poa pratensis. Application of lime significantly reduced the exchangeable fraction of Zn, whereas phosphate application had an accelerating effect on exchangeable Cu. With combined application of amendments, the plant metal concentration decreased by more than 40% for Cu and 70% for Zn, compared to soils receiving no amendments. Combined application of amendments, in conjunction with growth of Festuca and Poa, can be recommended for phytostabilising of Cu and Zn in moderately contaminated acid soils of southwest British Columbia.     

Phytoremediation of Metal-Contaminated Soil in Temperate Humid Regions of British Columbia,Canada

The suitability of five plant species was studied for phytoextraction and phytostabilisation in a region with temperate maritime climate of coastal British Columbia, Canada. Pot experiments were conducted using Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue), Helianthus annuus L (sunflower), Poa pratensis L (Kentucky bluegrass) and Brassica napus L (rape) in soils treated with three different metal (Cu, Pb, Mn, and Zn) concentrations. The bio-metric characters of plants in soils with multiple-metal contaminations, their metal accumulation characteristics, translocation properties and metal removal were assessed at different stages of plant growth, 90 and 120 DAS (days after sowing). Lolium was found to be suitable for the phytostabilisation of Cu and Pb, Festuca for Mn and Poa for Zn. Metal removal was higher at 120 than at 90 days after sowing, and metals concentrated more in the underground tissues with less translocation to the aboveground parts. Bioconcentration factors indicate that Festuca had the highest accumulation for Cu, Helianthus for Pb and Zn and Poa for Mn.     

Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 microM CuCl(2)) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 microg/l CuCl(2)). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C(18) reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.     

Allozyme Variation in the Rare Gulf Coast Endemic Polygonella macrophylla Small (Polygonaceae)

Abstract Polygonella macrophylla Small is a rare, perennial, primarily gynodioecious plant species endemic to a narrow zone of coastal sand pine scrub habitat along the Gulf of Mexico from Carrabelle, Florida, to Gulf Shores, Alabama (U.S.A.). The species is comprised of a crimson-red flowered form ("rubra"), known from only two populations at the eastern distributional limit of the species, and a white/pink flowered form ("alba") represented by several populations throughout the remainder of the range of the species. An electrophoretic investigation revealed that commonly used measures of gene diversity are very close to expected values in "rubra" given the combination of ecological attributes displayed by the species; however, gene diversity in "alba" is much lower than expected. Population differentiation is high, with estimates of the coefficient of gene differentiation ( G _ST ) indicating that more than 30% of the diversity is among populations. Lack of gene flow among populations, estimated to be at most 0.6 migrating individuals per generation for the species, is a likely cause of greater population differentiation. High inbreeding, detected both within populations and within the species, is suggestive of high rates of self-fertilization in hermaphroditic plants and/or biparental inbreeding in small populations.     

匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+ 胁迫的生长响应与阈限浓度

采用砂培法,研究了匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+胁迫的生长响应及阈限浓度,结果表明:种子萌发率随着4种重金属浓度的增加而下降。对株高的影响是当重金属浓度小于100mg/L时会促进株高生长,高于100mg/L则产生抑制作用。Cu2+显著抑制根系生长,并随浓度的增加抑制效应愈加显著;在Cu2+浓度为600mg/L时匍茎翦股颖的根长比对照下降了93.75%。Cu2+、Zn2+、Pb2+浓度小于200mg/L时会促进地上生物量的增加,但高于200mg/L时,地上生物量会随着3种重金属的增加而减少。Cu2+、Zn2+浓度小于100mg/L或Cd2+、Pb2+浓度小于200mg/L会增加叶绿素的含量,高浓度会降低叶绿素的含量;Cd2+在浓度为600mg/L时显著降低叶绿素含量,与对照相比,下降了43.55%。匍茎翦股颖生长的综合效应分析表明,匍茎翦股颖对Cu2+胁迫最敏感,具有较低的阈限浓度,而Zn2+胁迫对匍茎翦股颖的生长影响最小,阈限浓度相对较高。