135 resultados para heavy metal stress


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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.

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近年来植物重金属的耐性机制研究及抗重金属基因工程取得了很大进展。本文将来自于菜豆(Phaseolus vulgaris)的特异性重金属胁迫相关基因PvSR2 (Phaseolus vulgaris stress-related protein, PvSR)的cDNA序列克隆到大肠杆菌高效表达载体pBV221的PR PL启动子的下游,构建了原核表达载体pBV221-PvSR2。通过温度诱导,在大肠杆菌中成功地高效表达了PvSR2基因。经重金属(CdCl2)抗性检测,实验组比对照组有明显的抗性。 同时,将该基因克隆到植物转达化中间载体pCAMBEIA2301的花椰菜花椰病毒的35S启动子下游,利用根癌农杆菌(Agrobacterium tumefaciens)Ti质粒介导的遗传转化系统,成功地将该基因导入了烟草的基因组,获得了转基因植株。

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随着现代工业的发展,重金属污染日趋严重。重金属污染引发的环境和健康问题在许多国家都有报道,我国的重金属污染状况也不容乐观。土壤和水体中的重金属污染可以通过食物链进入人体,对人类健康造成很大的危害,如诱发癌症 和畸胎等。 植物修复是一种利用植物对重金属或有机污染物的超富集能力清除或减低污染的环境生物技术。植物修复的生物学机制的研究为这项技术走向实用化奠定了基础。植物修复近期的进展可能来自于可更有效地富集重金属的植物品种的选择、土壤条件的改善等;但长远看来,植物修复技术的巨大进步将取决于新的可更好地抵抗重金属或降解有机毒物的基因的鉴定和克隆,并通过转基因技术创造一批新的植物品种,如可迅速大量富集重金属的高生物量的用作环境净化的植物,以及可排拒重金属吸收的粮食、蔬菜和水果等作物。 本研究针对砷污染的植物修复机制,以超富集砷的凤尾蕨属植物——蜈蚣草为试材取得了如下进展: 1. 以从砷污染地区采集的蜈蚣草(Pteris vittataL.)为植物材料,利用抑制消减杂交(SSH)分离了经砷诱导处理与其对照间表达有差异的cDNA片段,以期得到与砷富集密切相关的基因。其中筛选到的一个cDNA片段与ABC transporter (ATP-binding cassette transporter)有较高的同源性。通过RACE方法对该基因进行了克隆,并进行了初步的结构和功能分析。结果表明所获得的PvABCTl (Accession No. AY496966)为一全长cDNA,长度为2165 bp,其中开读框架为1791 bp,编码597个氨基酸。该基因所编码的蛋白中含有2个ABC transporter特性结构域,1个ATP-binding cassette和2个ATP/GTP结合位点(P-loop),没有明显的跨膜区。 2. 对蜈蚣草在砷胁迫下PvABCT1基因的表达模式进行了研究。转录水平分析表明PvABCT1的表达受砷的诱导。进一步通过PvABCTl-GFP融合基因在洋葱细胞中的表达进行亚细胞定位,结果显示该基因可能定位于细胞质中。 3. 为了研究所克隆的PvABCT1基因的功能,本研究构建了PvABCT1的酵母表达载体,把该基因转入因ACR3基因缺失而对砷敏感的酵母突变株。酵母功能互补实验表明PvABCT1不仅不能与ACR3基因功能互补,反而使酵母对砷的敏感性增加,同时酵母细胞中的砷含量较未转化的酵母细胞增加。即在转入PvABCT1后,酵母细胞吸收了更多的砷。这暗示该基因与蜈蚣草中砷的高吸收有关。 针对食品重金属污染问题,本研究探讨了减低蔬菜对重金属吸收的方法及其 作用机理,取得了如下进展: 1.研究了钙离子和镧离子对镉离子胁迫下生菜种子萌发和植株生长的影响,结果表明在种子萌发时外施4 mM CaCI2或0.04 mg/L La(N03)3均可提高生菜对重金属镉的抗性。 2.通过检测0.5 mM CdCl2胁迫下生菜植株中的镉含量以及外施钙离子或镧离子后相应的镉含量,发现4 mM CaCl2可以增加镉胁迫下生菜植株中镉的积累;而0.04 mg/L La(N03)3可以降低镉胁迫下生菜植株中镉的积累。 3.对生菜中植物络合素合酶基因进行了克隆,通过RT-PCR分析以及植物络合素( phytochelatins,PCs)的检测,探讨了外施钙离子或镧离子对镉胁迫下生菜植株中植物络合素合酶基因在转录水平的表达量、植物络合素含量以及镉的积累三者之间的关系。结果表明:4 mM CaCl2可以提高镉胁迫下生菜植株中植物络合素合酶基因在转录水平的表达以及植物络合素的含量,增加镉的积累;而0.04 mg/L La(N03)3虽然同样可以提高植物络合素合酶基因在转录水平的表达以及植物络合素的含量,却能降低镉胁迫下生菜植株中镉的积累。这暗示外施钙离子可以促进用于重金属污染环境修复的植物对重金属的吸收,而外施镧离子可以用于降低叶菜类蔬菜中重金属镉的积累。

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The effects of cadmium (Cd2+) on growth status, chlorophyll (Chl) content, photochemical efficiency, and photosynthetic intensity were studied on Canna indica Linn. Plant specimens that were produced from a constructed wetland and precultivated hydroponically in 20 L of 1/10 Hoagland solution under greenhouse conditions for I week were exposed to cadmium in concentrations of 0, 0.4, 0.8, 1.6 and 3.2 mg L- Cd2+, respectively. The results show that leaves were injured in the Cd2+ solution by the third day of exposure and the injury became more serious with an increase in the applied heavy metal. Under 3.2 mg L-1 Cd2+ treatment, growth retardation, the decrease of chlorophyll content from 0.70 to 0.43 mg g(-1) FW, and a decrease in Chl a/b ratio from 2.0 to 1.2 were observed. Chl a was more sensitive than Chl b to Cd2+ stress. The decrease was the same with photochemical efficiency. Photosynthetic intensity decreased by 13.3% from 1.5X10(4) mumol m(-2)s(-1) CO2 in control to 1.3x10(4) mumol m(2)s(-1) CO2 in the treatment of 3.2 mg L-1. Because Canna species are used in heavy metal phytoremediation, these results show that C. indica can tolerate 0.4 to 0.8 mg L-1 Cd2+. Therefore, it is a potential species for phytoremediation of cadmium with some limitations only at higher concentrations.

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本论文以沈阳张士污灌区土壤为例,首次采用传统微生物生态学与现代微生物分子生态学相结合的研究方法系统地研究了污灌区长期重金属污染胁迫下原位农田土壤微生物特征。结果表明,虽然已经停止污灌十多年,张士灌区土壤耕作层(0~30 cm)仍然存在普遍的Cd污染,灌区土壤Cd含量高达1.75~3.89 mg kg-1。部分区域土壤Cd呈现向下迁移的趋势,且同时伴随有Cu、Zn复合污染。灌区土壤Cd含量较高时清水灌溉能降低土壤表层Cd含量,灌区土壤Cd含量下降到一定程度(约2 mg kg-1)后,清水灌溉对消除土壤表层Cd污染的作用消失。重金属元素中Cd对土壤微生物的影响最突出,在三个不同季节中土壤Cd与土壤微生物生物量(MBC)和微生物商(qM)呈显著负相关,与土壤微生物代谢商(qCO2)呈显著正相关。所检测的微生物指标中qM和qCO2与多种重金属元素呈显著相关性,可作为评价一定程度重金属污染的微生物指标。土壤营养元素(除P外)与微生物特征呈显著正相关性,土壤营养元素对微生物的刺激作用有可能在某种程度上掩盖了重金属对土壤微生物的负面影响。 用16S rDNA-PCR-DGGE方法,研究了不同浓度Cd胁迫下土壤Cd抗性细菌群落结构的动态变化,结果表明在Cd的胁迫下Cd抗性细菌多样性显著增加,不同土壤样品中Cd抗性细菌群落结构向相似的方向偏移,群落结构最终将可能趋向一致。Cd胁迫使敏感菌Pontibacter消失,而伯克氏菌(Burkholderia)、罗尔斯通氏菌(Ralstonia)、芽孢杆菌(Bacillus)和节杆菌(Arthrobacter)则富集成为优势菌。 从张士灌区Cd污染土壤中分离出32株Cd抗性细菌,研究了Cd抗性细菌和Cd抗性基因cadA的分布特征。这32株Cd抗性细菌分别归属于拟杆菌门(Bacteroidetes)(37.5%)、变形菌门(Proteobacteria) (37.5%)、放线菌门(Actinobacteria)(9.4%) 和厚壁菌门(Firmicutes)(15.6%)。在液体LB培养基中对Cd的抗性浓度都大于2 mmol L-1,对Zn抗性浓度介于5~13 mmol L-1。首次从Cetobacillus属的Cd抗性菌株S1基因组DNA中扩增出cadA基因的部分片断。在芽孢杆菌属(Bacillus)的4株菌N7,N9,N10和N11的基因组DNA中扩增出cadA基因的部分片断。序列分析结果表明这5株菌的cadA基因序列相似性为99%~93%,它们与坚强芽孢杆菌(Bacillus firmus) cadA 基因序列(M90750)相似性为94%~92%。系统发育分析结果表明这5株菌的cadA都与Bacillus firmus cadA 基因有着较近的亲缘关系。不同属的Cd抗性细菌间cadA基因的高度相似性揭示了cadA基因能在不同种属间转移的特性。

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本研究应用微波消解ICP-AES 法对62 个小麦品种及3 个地区土壤的锌铁硒含量进行了分析测定,发现不同小麦品种中微量元素含量差异很大,姊妹系间也存在差异。含铁量最高与最低的小麦品种铁含量相差29.68mg/kg。含锌量最高与最低的小麦品种锌含量相差46.70 mg/kg。含硒量最高与最低的小麦品种硒含量相差0.056 mg/kg。对不同地点的小麦及土壤中锌铁硒含量进行方差分析,发现双流和西昌两地种植小麦的铁含量和硒含量均有显著差异,西昌和荣县种植的锌含量有显著差异。在3 个地点中双流种植小麦硒含量最高,西昌种植小麦的铁和锌含量最高。 通过对小麦微量元素含量与土壤中微量元素含量进行了相关性分析,结果表明:小麦中的锌铁含量与土壤中的锌铁含量呈显著正相关,土壤中铁与锌含量呈极显著正相关,小麦中铁与锌含量也呈极显著正相关。随着土壤微量元素锌铁的提高,小麦中的锌铁元素含量同时提高,而且小麦对两种元素的吸收互相促进。土壤中的硒含量与锌铁含量呈负相关。小麦中硒含量也与锌铁含量也呈负相关。说明锌和铁与硒互相拮抗。小麦硒含量与土壤硒含量呈正相关,但不显著。表明土壤硒含量可以影响小麦硒含量,但不是决定因素,小麦硒含量与小麦自身因素有关。 对姊妹系G290(高硒含量)和G289(低硒含量)进行抗重金属胁迫和抗旱性实验发现,高硒品种G290的抗逆性优于低硒品种G289。 利用RAPD 技术对7 个姊妹系进行遗传差异分析发现,高硒材料G290出现了特异条带,分别标为1、2、3、4,其他姊妹系品种中未发现特异条带,回收4 条特异条带并连接转化,得到目的片段1、2、3 的重组子,进行测序。NCBI 中结果显示没有找到植物中的同源序列,说明特异序列可能是未发现的基因片段,推测可能与小麦硒含量有关,有待进一步研究。 以上研究结果,对小麦营养研究及功能性小麦的筛选和栽培具有指导作用。 In this study, we determinated the contents of zinc, iron, selenium in 62 wheat cultivars and soil samples of three regions by method of microwave digestion/ ICPAES,found that there was great difference of zinc, iron, selenium contents in different wheat cultivars as well as different sister lines. Iron content difference was 29.68 mg/kg between the highest-iron-content cultivar and the lowest one, and zinc content difference was 46.70 mg/kg , selenium content difference was 0.056 mg/kg. Anova analysis was made on contents of zinc, iron, selenium in wheat and soil samples of different locations, significant differences of Fe and Se contents were found between wheat in Shuangliu and Xichang, significant difference of Zn content was found between wheat in Xichang and Rongxian. Se content in wheat of Shuangliu was highest, Fe and Zn contents in wheat of Xichang were highest. Relativity analysis was made on three trace elements in Wheat and in soil, the result showed that there was significant positive correlation of zinc, iron content between in Wheat and in soil, as well as between Fe and Zn both in wheat and in soil. With the improving of Zn, Fe contents in soil, contents of Zn and Fe in wheat increased and absorption of Zn and Fe in wheat will mutual promote. Negative correlation of Se and Zn contents was found in wheat and soil, but not significant, that meant the antagonism of Se and Zn. Positive correlation of Se content in wheat and soil was found. High selenium content G290 and low selenium content G289 in sister lines were selected for heavy metal stress and drought resistance experiments, the result showed that the resistance of high-selenium-content cultivar was better than low selenium one. Analysis on genetic difference was made by RAPD, and specific bands were selected, marked 1,2,3,4, no more specific bands were found in other sister lines.4 bands were recovered, ligated to T-vector and transformed E.coli. Three recombinant plasmids were obtained and sequenced. NCBI Blast showed there was no homology with other plants. It implied that these fragments probably be new genes and maybe were related to selenium in wheat. It needs further research. This paper would be useful for the study of wheat nutrition as well as selection and cultivation of functional wheat.

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Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamysfarreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd2+, Pb2+ and Cu2+ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment. (c) 2007 Elsevier Inc. All rights reserved.

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卤虫(Artemia)是一种广温、耐高盐的小型甲壳动物,广泛分布于内陆盐湖和沿海盐田中。卤虫的无节幼体作为重要的蛋白优质饵料,被广泛的应用于水产养殖生产。卤虫具有特殊的生物学特性,是研究甲壳动物胚胎发育的良好的实验材料,同时也是一种研究动物抗逆机制的模式动物。卤虫有卵生和卵胎生两种繁殖后代的方式,当环境条件适宜时,卤虫倾向于采取卵胎生方式,即直接产生无节幼体;而在恶劣的环境条件下,卵生方式占主要地位,产生处于滞育状态的、具有复杂外壳的休眠卵。卤虫的滞育卵具有独特的生物学特性和特殊的生理生化特点。其发育停滞,细胞分裂停止,酶活力下降,代谢活动受到抑制并可耐受各种极端恶劣环境,如缺氧、低温、紫外线、干燥等。即使在最适的环境中滞育卵的孵化率也很低,只有受到某些特定的非生物信号的刺激才自能终止这种滞育状态,恢复生理代谢;当环境条件适宜时,能够继续发育孵化成无节幼体。因此,卤虫的滞育卵在卤虫的整个生活史中占有重要的地位。另一方面,卤虫是极端环境生物,能够抵抗各种恶劣环境胁迫刺激,因此是研究抗逆机理的良好的实验动物。 本论文利用蛋白质组学技术,研究了卤虫滞育卵及滞育卵发育过程中的蛋白质组表达情况,并研究了卤虫幼体在重金属刺激后蛋白表达的变化情况。得到如下结果: 建立了中华卤虫滞育卵可溶性总蛋白的双向凝胶电泳对照图谱。在pH 4–7、分子量10-100 kDa范围内,检测到约 233个蛋白点,并利用高效液相色谱-质谱联用(LC-ESI-MS/MS)技术鉴定了其中的48个丰度较大及感兴趣的蛋白点,根据这些蛋白的生物学功能进行分类,功能类别包括细胞防御蛋白、抗氧化蛋白、细胞骨架蛋白、代谢相关蛋白等。在卤虫滞育卵中共分离鉴定到6个分子量和等电点存在差异的小热休克蛋白p26的异构体,生物信息学分析表明该蛋白有三种不同的功能位点,分别是蛋白激酶C磷酸化位点,Casein 激酶II磷酸化位点及 N-myristoylation 位点。 采用低温脱水的方法对滞育卵进行激活刺激,并对活化卵和滞育卵蛋白表达图谱进行了对比分析。结果表明对卤虫滞育卵的激活刺激引起了其蛋白表达的明显变化。活化卵图谱中蛋白点总数比滞育卵中明显增多,特别是在pI<5.5范围内。约70个蛋白点在激活刺激后上调表达,包括部分只在激活卵中表达的蛋白;25个下调表达,包括部分只在滞育卵中表达的蛋白;其余约60%(占滞育卵蛋白点数目百分比)的蛋白点表达量基本恒定。热休克蛋白家族、抗氧化蛋白家族成员等蛋白变化明显,小热休克蛋白p26、小热休克蛋白ArHsp21蛋白以及过氧化物还原酶异构体在激活卵中特异表达。 活化卵孵化过程中不同发育时期的蛋白表达又呈现出不同的特点,分别在孵化后6h、12h、18h和24h的蛋白质组学图谱上检测到267、285、195和210个蛋白点。孵化后6h和12h休眠卵蛋白表达个数相对较多,与胚胎发育过程中的器官发生和剧烈的形态变化相适应;孵化后18h和24h休眠卵蛋白表达明显下降,部分蛋白的表达关闭,部分蛋白开始富集表达。 利用双向凝胶电泳技术分析了中华卤虫幼体受到急性硫酸铜刺激后的蛋白表达变化情况。通过图谱对比分析,检测到了5mM硫酸铜刺激24h后,卤虫幼体中14个差异表达的蛋白点。利用LC-ESI-MS/MS技术鉴定了其中的7个蛋白,其中3个蛋白上调表达,分别是热休克蛋白70(7.5倍), 肌动蛋白(2.3倍)和伴侣分子亚基1(3.0倍)。3个蛋白下调表达,分别是:精氨酸激酶(2.8倍), 延伸因子2 (2.0倍) 和富含甘氨酸蛋白(2.0倍)。硫酸铜刺激后特异表达的一个蛋白被鉴定为过氧化物还原酶(Peroxiredoxin,Prx)。根据质谱检测提供的蛋白肽段信息和其他生物过氧化物还原酶保守氨基酸序列设计简并引物,结合RACE技术,从中华卤虫幼体中克隆到了过氧化物还原酶基因,该基因的cDNA全长为756个碱基,其中开放阅读框为594个碱基,编码198个氨基酸,其蛋白理论分子量为22.0 kDa,理论等电点为6.98。多序列比对结果显示中华卤虫Prx基因的推导氨基酸序列与美国卤虫和中国对虾的同源性高达98%和94%。实时荧光定量PCR结果显示,硫酸铜刺激后,该基因在卤虫无节幼体中的转录水平明显升高,在24h达到正常水平的3.0倍。

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A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H2O2, the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (I h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24 h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. (c) 2006 Elsevier GmbH. All rights reserved.

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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.

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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.