Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity.Since phytochelatins can effectively chelate heavy metals,it was hypothesized that S.salsa possessed a phytochelatin synthase(PCS) gene.In the present study,the cDNA of PCS was obtained from S.salsa(designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends(RACE).A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides,encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain.A similarity analysis suggested that SsPCS shared up to a 58.6%identity with other PCS proteins and clustered with PCS proteins from eudicots.There was a new kind of metal ion sensor motif in its C-terminal domain.The SsPCS transcript was more highly expressed in elongated and fibered roots and stems(P<0.05) than in leaves.Lead and mercury exposure significantly enhanced the mRNA expression of SsPCS(P<0.05).To the best of our knowledge,SsPCS is the second PCS gene cloned from a halophyte,and it might contain a different metal sensing capability than the first PCS from Thellungiella halophila.This study provided a new view of halophyte PCS genes in heavy metal tolerance.

Enhanced Cadmium Accumulation in Transgenic Tobacco Expressing the Phytochelatin Synthase Gene of Cynodon dactylon L

Bermudagrass （Cynodon dactylon L. cv. Goldensun） is highly resistant to and accumulates large amounts of cadmium （Cd）. A phytochelatin synthase （PCS） cDNA （CdPCS1） was isolated from this grass by rapid amplification of cDNA ends. The putative CdPCS1 protein shared a high homology with PCS from other plants, with 79% homology at the N-terminal and 47% homology at the C-terminah However, 16 Cys residues were found at the C-terminal of CdPCS1, and among these residues, three positions were different from other PCS proteins. Semiquantitative reverse transcription-polymerase chain reaction analysis showed that Cd stress induced CdPCS1 expression in both roots and leaves in Bermudagrass. We verified that CdPCS1 plays an important role in Cd tolerance in yeast cells by expressing the gene in ABDE1, a Cdsensitive mutant. CdPCS1 was then introduced into tobacco plants. The phytochelatin level in some transgenic tobacco lines increased 3.88-fold more than in wild type plants and Cd accumulation in these transgenic plants was enhanced 3.21-fold accordingly. The results suggested that CdPCS1 could be used as a gene element for phytoremediation in the future.

RNA Interference-mediated Silencing of Phytochelatin Synthase Gene Reduce Cadmium Accumulation in Rice Seeds

Phytochelatins （PCs） play an important role in heavy metal resistance and accumulation. To reduce the accumulation of cadmium （Cd） in rice seeds, the expression of phytochelatin synthase （PCS） gene OsPCS1 was suppressed by RNA interference （RNAi）. A hairpin construct of a PCS fragment was designed in the pRNAi-OsPCS1 under the control of ZMM1, a seed-specific promoter from maize. The construct was introduced into rice （japonica） through Agrobacterium tumefaciens. The RNAi rice plantlets were selected and cultivated in pots exposured to 10 mg/kg Cd. The transcriptional level of OsPCS1 declined in seeds of some RNAi rice compared to the wild type. As a result Cd accumulation was reduced by about half in the seeds of RNAi rice. As expected, no apparent difference of growth appeared between RNAi and wild-type plants. The results suggest that this new approach can be used to control heavy metal accumulation in crops.

Effects of Interaction Between Cadmium and Plumbum on Phytochelatins and Glutathione Production in Wheat （Triticum aestivum L.）

: Phytochelatins (PCs) may function as a potential biomarker for metal toxicity. However, less attention has been paid to the effects of metal interactions on the production of PCs and glutathione (GSH), the most prominent cellular thiol. In the present study, the effects of interactions between cadmium (Cd) and plumbum (Pb) on the production of PCs and GSH were monitored over a period of 14 d in wheat (Triticum aestivum L.) tissues. The results showed that combination of Cd and Pb led to synergistic growth inhibition in wheat. Exposure to Cd or Pb increased levels of PCs in a concentration-, tissue-, and time-dependent manner. Cadmium was more effective that Pb in increasing PCs production. Compared with the effects of Cd or Pb alone on the production of PCs, the combination of Cd and Pb acted synergistically, resulting in an enhanced production of PCs. Cadmium also stimulated GSH production in a concentration-, tissue-, and time-dependent manner. However, Pb had no obvious effects on GSH levels. The combination of Pb and Cd antagonized GSH production over the course of the growth period. The results of the present study suggest that metal interactions should be considered in the application of PCs and GSH as potential biomarkers for the evaluation of metal toxicity.

镉污染土壤的植物修复研究进展

工业发展中产生的重金属污染土壤是环境污染中的一个重要问题。对Cd含量超标的土壤进行植物修复是一种环境友好、无二次污染和修复比较彻底的优势策略。由于土壤重金属污染源的特异性和复杂性,以及受到周围生态环境和物种的限制,在制定植物修复策略时需要多方面综合考虑。文章以植物修复为主,按植物功能分为观赏植物、经济植物、能源植物、药用植物、粮食作物和蔬菜植物等,比较分析富集能力,并联合动物修复和微生物修复对Cd污染土壤的修复展开论述,分析利弊,以期为植物修复Cd污染土壤提供策略,并为合理制定修复方案提供参考。

The importance of glutathione and phytochelatins on the selenite and arsenate detoxification in Arabidopsis thaliana

We investigated the role of glutathione（GSH） and phytochelatins（PCs） on the detoxification of selenite using Arabidopsis thaliana. The wild-type（WT） of Arabidopsis thaliana and its mutants（glutathione deficient Cad 2–1 and phytochelatins deficient Cad 1–3） were separately exposed to varying concentrations of selenite and arsenate and jointly to both toxicants to determine their sensitivities. The results of the study revealed that, the mutants were about 20-fold more sensitive to arsenate than the WT, an indication that the GSH and PCs affect arsenate detoxification. On the contrary, the WT and both mutants showed a similar level of sensitivity to selenite, an indication that the GSH and PCs do not significantly affect selenite detoxification. However, the WT is about 8 times more sensitive to selenite than to arsenate, and the mutants were more resistant to selenite than arsenate by a factor of 2. This could not be explained by the accumulation of both elements in roots and shoots in exposure experiments. The co-exposure of the WT indicates a synergistic effect with regards to toxicity since selenite did not induce PCs but arsenic and selenium compete in their PC binding as revealed by speciation analysis of the root extracts using HPLC–ICP–MS/ESI–MS. In the absence of PCs an antagonistic effect has been detected which might suggest indirectly that the formation of Se glutathione complex prevent the formation of detrimental selenopeptides. This study, therefore, revealed that PC and GSH have only a subordinate role in the detoxification of selenite.

Root cell walls and phytochelatins in low-cadmium cultivar of Brassica parachinensis

‘Lvbao-701’ is a cultivar of Chinese flowering cabbage(Brassica parachinensis) that exhibits low cadmium(Cd) accumulation and high Cd tolerance.In this study, this cultivar was compared with a high-Cd accumulating cultivar, ‘Chixin-4’, to characterize the mechanisms influencing Cd accumulation in B. parachinensis. Root cell walls were isolated by dissolving the cytoplasm with an organic solvent, and root Cd and phytochelatin(PC) contents were analyzed. In addition, a PC synthase gene fragment was cloned and expressed under Cd stress conditions. The proportions of Cd bound to root cell walls were higher in the ‘Lvbao-701’ plants(68.32%–76.80%) than in the ‘Chixin-4’ plants(35.36%–54.18%) after exposure to Cd stress. The proportions of Cd bound to root cell walls measured using cell walls isolated with a non-destructive method were higher than those obtained using a conventional method that required grinding and centrifugation. Exposure to Cd stress induced the PC production and resulted in higher PC contents in the ‘Lvbao-701’ plants than in the ‘Chixin-4’ plants. Cloning and expression analysis of a B. parachinensis PC synthase cDNA fragment indicated that PC synthase gene expression was induced by Cd and occurred mainly in the roots of both ‘Lvbao-701’ and ‘Chixin-4’ plants. However, the PC synthase gene expression level was higher in the‘Lvbao-701’ roots than in the ‘Chixin-4’ roots. Therefore, a higher abundance of Cd in the root cell walls of ‘Lvbao-701’ and up-regulated PC production in the roots are probably the main reasons why ‘Lvbao-701’ exhibits lower Cd translocation to the shoots and higher tolerance to Cd than ‘Chixin-4’.

Synergistic defensive mechanism of phytochelatins and antioxidative enzymes in Brassica chinensis L. against Cd stress

Brassica chinensis L. was chosen and exposed to different concentrations of Cd exposure to evaluate its Cd-accumulating capacity and its potential cellular defensive mechanisms. Cd accumulation in the shoots and roots of B. chinensis was up to 1348.3±461.8 and 3761.0±795.0 mg per killogram of dry weight, respectively, under 200 μmol/L of Cd exposure. Increasing Cd accumulation in the plant was accompanied by rapid accumulation of phytochelatins (PCs), and the sequestration of Cd by PCs provided a primary cellular mechanism for Cd detoxification and tolerance of B. chinensis. Furthermore, malondialdehyde formation, hydrogen peroxide content and antioxidative enzyme activities such as superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase were observed in the shoots of Cd-stressed B. chinensis. Increasing enzyme activities in response to concentrations of 5 to 50 μmol/L Cd showed an efficient defense against oxidative stress, suggesting that the antioxidative system was a secondary defensive mechanism. These resulted in reduced free Cd damage and enhanced Cd accumulation and tolerance. Glutathione plays a pivotal role in these two detoxification pathways. In general, these results suggested that PCs and the antioxidative system are synergistic in combatting Cd-induced oxidative stress and that they play important roles in Cd detoxification of B. chinensis, and also give a deep understanding of the natural defensive mechanisms in plants under heavy metal stress.

植物对镉胁迫响应的研究进展

总结了近年来植物对一重要环境污染物镉的反应研究报道。探讨了镉对植物的各种毒性效应 ,并论述植物对镉胁迫采取的相应防御机制 ,如络合 ( PC、MT)、应激 (应激乙烯、应激蛋白 ) ,及额外防御机制 ,如细胞壁固定化、原生质膜排除、区域化。

分子生物标志物在污染环境早期诊断和生态风险评价中的应用

介绍了抗氧化防御系统、应激蛋白HSP70、活性氧(ROS)以及植络素(PCs)等作为分子水平上的生物标志物,在水和土壤环境污染早期诊断和生态风险评价中的研究现状及最新进展,以及污染物胁迫下活性氧的产生及对机体的氧化损伤机理,并展望了分子生物标志物在污染环境早期诊断和生态风险评价中的发展方向.

大麦中镉的亚细胞分布和化学形态及PCs合成的基因型差异

为探明不同基因型大麦对镉耐性和转运能力差异，选取大麦耐镉品种华10118和敏感品种华2255进行水培试验，比较不同镉处理下大麦根系和地上部中镉的亚细胞分布和化学形态的差异，并研究了根系和叶片中PCs含量对镉转运能力的影响。结果表明：华10118对镉的转运能力较差，地上部镉含量较低；镉在两个大麦品种中主要分布于细胞壁和胞液中，基本各占35％-44％，细胞器占5％-26％，华2255的细胞器组分中镉含量显著大于华10118，可能是华2255耐镉性差的原因。两个基因型大麦根系中的镉以醋酸提取态为主，而地上部中以NaCl提取态最多；华2255的乙醇提取态、去离子水提取态和NaCl提取态的镉含量较高，可能是其对镉转运能力较强和耐受性差的原因。华2255根系和叶片中的PC2和PC3含量都显著高于华10118，根系合成较多的Pc：和PC，有助于镉从根部向地上部的转运。

Cr^(3+)胁迫对青菜中植物络合素含量及AsA-GSH代谢关键酶活性的影响

采用不同浓度Cr3+处理,测定不同耐铬性青菜品种叶片中抗坏血酸-谷胱甘肽(AsA-GSH)代谢关键酶活性及植物络合素含量等生理指标。结果表明,随着处理浓度的增加,两个青菜品种叶片中植物络合素及抗氧化剂含量呈增加趋势。高浓度Cr3+(250mg·L-1)处理下,耐铬性强的矮抗青品种叶片中超氧阴离子(O-2·)含量与对照相比增幅较小,耐铬性差的绿秀品种则增幅较大;耐铬性强的矮抗青叶片中还原型抗坏血酸(AsA)、还原型谷胱甘肽(GSH)含量显著高于对照(P<0.05),而绿秀品种则略增,差异不显著(P>0.05);耐铬性强的矮抗青品种叶片中脱氢抗坏血酸(DHA)、氧化型谷胱甘肽(GSSG)含量分别是对照的1.87、1.72倍,低于耐性差的绿秀品种(1.98、1.94倍)。随着铬处理浓度的增加,AsA-GSH关键酶活性呈先增后降趋势。高浓度Cr3+处理下,耐铬性强的矮抗青品种叶片中植物络合素(PCs)含量、抗坏血酸过氧化物酶(APX)及脱氢抗坏血酸还原酶(DHAR)活性分别比对照增加了30.9%、38.9%、45.2%,而耐性差的绿秀分别增加了11.5%、30.5%、43.0%。综合分析表明,矮抗青在Cr3+处理下具有较高的AsA-GSH代谢关键酶活性和非酶物质含量,可能是不同青菜品种对Cr3+耐受性差异的内在生理原因。

铜、锌离子抗性菌筛选及重金属作用下富集特性研究

从湖南临乡桃林矿区土壤中分离到一株高抗铜、锌菌株,经26S rRNA D1/D2鉴定为Aspergillus aculeatus.该菌株单抗铜400 mg/L、锌800 mg/L.优化条件下:30℃,pH值5.0,起始离子浓度50 mg/L,摇床转速120 r/min,培养时间120 h,菌体对铜、锌离子的吸附率为54%和60%.铜、锌离子浓度50 mg/L时,菌体内谷胱甘肽含量达到最大值分别为0.98和0.88 mg/g;而正常菌体内只有0.04 mg/g.可初步认为谷胱甘肽的增加缓解了铜、锌离子对Aspergillus aculeatus的氧化损伤.

植物螯合肽及其在抗重金属胁迫中的作用

植物螯合肽 (PCs)广泛存在于植物体中 ,与植物抗重金属胁迫关系密切。植物螯合肽及其复合物是一类富含半胱氨酸的低分子量化合物。现有研究证明 ,PCs由谷胱甘肽 (GSH)为底物的酶促反应合成 ,其合成受相关基因的调控 ,从模式植物拟南芥的突变体中已分离到与 PCs合成有关的几个基因。植物螯合肽首先与重金属离子结合形成低分子量 (LMW)复合物 ,以此形态经由细胞质进入液泡后 ,再与一个分子的植物螯合肽结合 ,形成对植物组织毒性较小的高分子量 (HMW)复合物 ,从而达到缓解重金属对植物的危害作用。就植物螯合肽及其复合物的结构、生物合成、基因调控及重金属解毒机理等进行了综述 。

铬胁迫对青菜(Brassica chinensis L.)植物螯合肽含量及抗氧化特性的影响

选用不同耐铬胁迫青菜品种春油1号和青伏令,采用不同浓度Cr3+处理,测定青菜抗氧化酶活性及植物螯合肽含量等生理指标。结果表明,随着Cr3+处理浓度升高,两种青菜叶片抗氧化酶活性、酸溶性SH、植物螯合肽(PCs)及谷胱甘肽(GSH)含量均呈增加趋势(P<0.05),高浓度Cr3(+300 mg.L-1)处理下,耐铬胁迫较弱的青伏令叶片SOD、POD、CAT活性分别比对照增加了24.2%、41.7%、21.4%,耐铬胁迫较强的春油1号分别比对照增加了37.2%、70.7%、43.0%,并且PCs含量是对照的3.17倍,明显高于耐铬胁迫较弱品种青伏令(1.71倍)。Cr3+处理下耐铬胁迫较强的青菜品种春油1号表现出相对较高的抗氧化酶活力及非酶物质含量,不同品种青菜对铬胁迫的生理差异较大。

硫对镉胁迫下小白菜叶片AsA-GSH循环和植物络合素含量的影响

采用水培试验研究了不同镉、硫水平对小白菜叶片中抗坏血酸-谷胱甘肽(AsA-GSH)循环和植物络合素含量的影响。结果表明,适度增硫降低了镉胁迫下小白菜叶片中过氧化氢(H2O2)和丙二醛(MDA)含量,提高了AsA-GSH循环中关键酶的活性、抗氧化物质和植物络合素的含量。低镉(5μmol·L-1)水平下,随着硫浓度的增加,小白菜叶片中H2O2的含量显著下降,MDA含量先降后升,谷胱甘肽还原酶(GR)活性、抗氧化物质及植物络合素含量持续升高,抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)和单脱氢抗坏血酸还原酶(MDHAR)及AsA含量呈先升后降的趋势,且均在3mmol·L-1硫处理达到峰值。高镉(50μmol·L-1)水平下,随着硫浓度的增加,小白菜叶片中H2O2和MDA含量均先降后升,AsA-GSH循环关键酶活性、抗氧化物质及植物络合素含量均呈先升后降趋势,且多数在2mmol·L-1硫处理达到峰值。研究表明适度增硫处理能维持小白菜叶片AsA-GSH循环快速有效地运转,增加植物络合素的合成,保持较强的抗氧化物质的再生能力和重金属螯合解毒能力,对降低镉胁迫伤害具有重要作用。但是过高SO2-4离子浓度存在一定的盐胁迫毒害效应,因而影响其对小白菜镉生物毒性的降低作用。

小麦在铜、镉胁迫下体内含巯基物质对解毒机制的研究

将小麦幼苗分别放在含有100μmol／L Cu2＋的培养液和100μmol／LCd2＋的培养液中培养，观察其体内重金属含量、丙二醛（MDA）、植物螯合肽（Pc）和谷胱甘肽（GSH）的变化。试验结果表明，在铜、镉胁迫下，小麦体内重金属含量逐渐增加，尤以根中增加明显，约75倍，而叶中增加较少，为2～5倍；无论是在根中还是在叶中，镉的含量都高于铜的含量；铜、镉胁迫下小麦叶片膜脂过氧化程度都高于根部无论是铜胁迫还是镉胁迫下，小麦体内叶片的GSH含量均高于根部，而PC含量均低于根部。

大蒜植物络合素合酶基因转化对酵母重金属抗性的提高(英文)

重金属污染是全球面临的亟待解决的生态问题。利用植物对重金属的富集作用来清除环境重金属污染即植物修复已成为重要的环境生物技术之一。这一技术的长远发展有赖于在重金属富集或耐受中起关键作用的基因的克隆和应用。植物络合素是植物体内一类重要的对重金属起螯合作用的多肽, 其合成受植物络合素合酶的催化。该文取得了如下研究结果:1)通过原子吸收测定表明,在大蒜(Allium sativum)的根部可以积累3 000 mg·kg-1的重金属镉;2)将克隆的大蒜植物络合素合酶基因(AsPCS)置于酵母表达启动子之下,构建酵母表达载体,并将其分别转入了因CUP1和acr3基因缺失而对重金属镉和砷敏感的酵母突变体菌株后,发现来自大蒜的AsPCS基因的表达使酵母CUP1缺失菌株对镉的耐受性提高了4倍, acr3缺失菌株对砷的耐受性提高了两倍;3)表达AsPCS基因酵母的生长模式证实了AsPCS基因的表达是酵母对重金属耐受性提高的原因。这些结果暗示, 大蒜植物络合素合酶基因在大蒜对重金属的抗性及大蒜根部对镉的积累中起关键作用,可作为重要的基因元件应用到修复污染的植物基因工程中。

镉胁迫下不同大豆品种各器官中镉和非蛋白巯基物质的动态变化

【目的】探讨不同大豆品种各器官镉和非蛋白巯基物质的动态变化,揭示巯基物质在大豆镉抗性和积累中的作用。【方法】在镉(Cd)污染的土壤中种植镉抗性和籽粒积累不同的大豆品种中黄24和华夏3号,采集不同生育时期的根、茎和叶,分析各品种的Cd抗性指标(根系和地上部鲜质量)、Cd积累指标(各器官Cd浓度)和非蛋白巯基物质[总非蛋白巯基肽(NPT)、谷胱甘肽(GSH)和植物螯合肽(PC)]的浓度变化。【结果】在10 mg·kg-1的Cd胁迫下,中黄24不同时期Cd抗性指标明显低于华夏3号,而中黄24初花期后各器官Cd浓度均显著地高于华夏3号。Cd胁迫下2个品种随发育进程根系中NPT、GSH和PC浓度上升,而叶片中则下降;敏感品种中黄24各器官中巯基物质对Cd胁迫响应比抗性品种华夏3号更显著。关联分析发现,大豆根部巯基物质浓度与各器官Cd浓度呈正相关,且以成熟期最为显著,而初花期后地上部的PC与各器官Cd浓度呈负相关。【结论】在大豆不同生育期不同器官中Cd和非蛋白巯基物质浓度变化复杂,非蛋白巯基物质在大豆抵抗Cd胁迫中扮演多种角色。

维管植物的镉毒和耐性机制

本文讨论了影响土壤中镉有效性的因素和植物对镉的吸收与转运。从植物的生长、光合作用、气孔功能、水分关系、酶活性等方面阐述了镉对维管植物的毒害机制 ;并对近年来有关维管植物通过体内镉的积累与分隔和镉结合复合物产生的镉耐性机制的研究作一综述。