伴矿景天植物螯合肽合酶基因的克隆及功能分析

重金属超积累植物由于长期生长在高浓度的重金属环境中,使得经由植物螯合肽（phytochelatins, PCs）解毒途径来应对重金属毒害代价高昂。我们从Zn/Cd超积累植物伴矿景天（Sedum plumbizincicola）中克隆了植物螯合肽合酶（phytochelatin synthase, PCS）基因SepPCS。该基因在裂殖酵母和拟南芥中表达后都具有PCS活性,而且能够互补它们的PCs缺失突变体的Cd敏感表型。SepPCS在伴矿景天中的表达受到高浓度Cd处理的诱导。与其近亲非超积累生态型东南景天（S. alfredii）相比,虽然伴矿景天地上部PCs与Cd的摩尔比远低于东南景天,但是在高浓度Cd处理时PCs含量以及PCs与Cd的摩尔比急剧增加。我们推测在伴矿景天应对Cd毒害的过程中, PCs起到一定的作用,并且在高浓度Cd胁迫时地上部PCs依赖的解毒作用有所加强。     

不同类型污染土壤中赤子爱胜蚓对伴矿景天重金属吸收的影响初报

伴矿景天Sedum plumbizincicola是我国发现和报道的镉/锌(Cd/Zn)超积累植物,在土壤Cd污染修复方面已开展实际应用。由于超积累植物伴矿景天在不同类型土壤下的生长能力以及对镉锌的去除效果存在较大差异,因此需引入强化修复技术为植物修复提供辅助作用。作为大型土壤动物,蚯蚓对植物生长的促进作用已有较多研究,但其对伴矿景天生长和重金属吸取效率的影响则鲜有报道,为探究赤子爱胜蚓对不同类型土壤种植下的伴矿景天是否具有强化修复效应,以及不同类型土壤下的强化修复效应差异,设计以下盆栽试验。通过在常湿淋溶土(Perudic Luvisols)、水耕人为土(Stagnic Anthrosols)、湿润雏形土(Udic Cambisols)3种土壤上种植伴矿景天、引入赤子爱胜蚓Eisenia foetida,探究赤子爱胜蚓对伴矿景天生长及Cd/Zn吸收性的影响。选取Cd有效性较高、修复潜力较大的水耕人为土(Stagnic Anthrosols)进行第二季盆栽修复试验。第一季修复结果显示,在酸性的常湿淋溶土中,添加赤子爱胜蚓使伴矿景天地上部生物量较对照处理增加了106%,Cd和Zn吸收量分别提高了72.0%和36.0%,且蚯蚓结合伴矿景天的处理修复后土壤Cd有效性进一步降低,其余两种土壤仅添加蚯蚓无强化修复效应;第二季结果显示,同时添加秸秆和蚯蚓,可强化中性的水耕人为土上种植的伴矿景天生长,增大植物地上部生物量和Cd/Zn吸收量。结果表明,添加蚯蚓可增强伴矿景天在常湿淋溶土中的养分吸收,提高生物量,以此强化其修复效应。在水耕人为土中,外加秸秆可作为蚯蚓强化伴矿景天修复的配套技术。     

植物螯合肽的转运与功能研究进展

植物螯合肽（phytochelatins,PCs）是由植物螯合肽合酶催化谷胱甘肽合成的一类生物小分子,结构式为（γ-Glu-Cys）n-Gly（n=2-11）,在真菌和高等植物耐受重金属胁迫机制中具有重要作用。近年来,人们在Pc介导重金属脱毒害的分子机理研究上取得了重要进展,发JLSpHMT1和SpABC2是PC在裂殖酵母中介导重金属液泡区室化的主要转运蛋白,鉴定了拟南芥液泡膜PC转运蛋AtABCC1和AtABCC2。此外,PCs也可能在超积累植物细胞内对重金属脱毒害具有重要功能。     

巨大芽孢杆菌对伴矿景天修复镉污染农田土壤的强化作用

伴矿景天(Sedum plumbizincicola)是一种Cd/Zn超积累植物,常用于Cd污染土壤的植物修复。巨大芽孢杆菌(Bacillus megaterium)是一种溶磷型细菌,既可以促进植物生长,也可以提高土壤重金属生物有效性,对重金属污染土壤植物修复具有强化作用。本研究采用盆栽试验方法,分析了巨大芽孢杆菌不同接种量(10～60 mL)对伴矿景天修复Cd污染农田土壤效率的影响。结果表明: 在Cd污染农田土壤中接种巨大芽孢杆菌可以提高土壤中Cd的活性,土壤有效态Cd含量较对照(CK)增加15.0%～45.0%。与CK相比,巨大芽孢杆菌提高了伴矿景天地上和地下部的生物量,增幅分别为8.7%～66.7%和13.6%～81.8%,并显著增加了伴矿景天地上部的Cd含量,增幅在29.2%～60.4%。在种植伴矿景天并接种巨大芽孢杆菌条件下,土壤Cd去除率在26.7%～42.9%。这说明接种巨大芽孢杆菌可以促进伴矿景天的生长,增加其Cd含量,从而提高Cd污染农田土壤的修复效率。     

镉超积累植物及植物镉积累特性转基因改良研究进展

植物提取修复技术是一项既经济又环保的土壤镉(Cd)污染修复技术,该技术的关键是筛选Cd超积累植物或利用基因工程手段改良植物以提高其Cd积累能力。人们已发现遏兰菜等7种Cd超积累植物及美人蕉等潜在的Cd超积累植物。还发现了许多与Cd耐受和积累能力有关的基因:(1)编码与Cd积累、耐受有关酶的基因,如细菌中的ACC(1-aminocyclopropane-1-carboxylic acid),植物中的PCS(Phytochelatin Synthase)基因;(2)编码金属结合蛋白的基因:MT(Metallothionein)、转运蛋白(P-type ATPase、ABC型转运器)基因;(3)其它相关基因:Hvhsp17、PvSR2(Phaseolus vulgaris stress-related gene number 2)等。并将其中的一些基因转入到其它生物中,提高了其对Cd的耐受性和积累量,为实现Cd污染土壤修复的目标奠定基础。     

改良剂对酸性土壤上伴矿景天铝毒缓解作用及镉锌吸收性的影响

为探究不同改良剂对酸性土壤铝(Al)胁迫条件下镉(Cd)锌(Zn)超积累植物伴矿景天Sedum plumbizincicola生长以及镉和锌吸取修复效率的影响,分别添加不同种类改良剂(钙镁磷肥(CMP)、MgCO3、KH2PO4)和不同浓度CMP进行温室盆栽试验。结果表明,CMP能够一定程度上提高土壤pH值并降低土壤交换性Al的浓度,MgCO3能够显著提高土壤pH值和降低土壤交换性Al的浓度,KH2PO4能够降低土壤中交换性Al浓度但未改变土壤pH值。施用适量的CMP(9.39 mg/kg)能够提高伴矿景天生物量和Cd、Zn吸取修复效率,用量过高会抑制伴矿景天生长和Cd、Zn修复效率;施用MgCO3可增大伴矿景天生物量和Cd、Zn修复效率,施用KH2PO4反而抑制了伴矿景天生长。酸性土壤上施用适量的CMP和MgCO3能够缓解伴矿景天的铝毒作用,维持较高的重金属吸收效率。     

伴矿景天-水稻轮作及磷修复剂对水稻锌镐吸收的影响

采用盆栽试验,将锌镉超积累植物伴矿景天与镉低积累水稻中香1号轮作种植于重金属污染土壤,并向土壤添加钙镁磷肥和磷矿粉,研究两种磷修复剂对伴矿景天和锡低积累水稻生长及地上部重金属积累性的影响.盆栽试验结果表明,在轻污染土壤上施用50g"kg-'磷矿粉时伴矿景天地上部的Zn,Cd吸收量分别达到每盆11.5和0.79 mg,效果好于施用4g·kg(-1)钙镁磷肥处理.在重金属污染土壤上种植伴矿景天使后茬水稻地上部Zn、Cd浓度上升,但钙镁磷肥的施用显著降低了水稻体内的Zn,Cd积累量.种植伴矿景天后添加钙镁磷肥稳定调控剂对土壤中水溶态及NH4 OAc提取态Zn、Cd的稳定效果明显优于磷矿粉,且在高污染土壤上效果更佳.田间试验结果显示,施用钙镁磷肥不仅可增加水稻产量,且可一定程度上降低水稻地上部的Zn、Cd吸收量.     

伴矿景天-水稻轮作及磷修复剂对水稻锌镉吸收的影响

采用盆栽试验,将锌镉超积累植物伴矿景天与镉低积累水稻中香1号轮作种植于重金属污染土壤,并向土壤添加钙镁磷肥和磷矿粉,研究两种磷修复剂对伴矿景天和镉低积累水稻生长及地上部重金属积累性的影响.盆栽试验结果表明,在轻污染土壤上施用50 g·kg^-1磷矿粉时伴矿景天地上部的Zn、Cd吸收量分别达到每盆11.5 和0.79 mg,效果好于施用4 g·kg^-1钙镁磷肥处理.在重金属污染土壤上种植伴矿景天使后茬水稻地上部Zn、Cd浓度上升,但钙镁磷肥的施用显著降低了水稻体内的Zn、Cd积累量.种植伴矿景天后添加钙镁磷肥稳定调控剂对土壤中水溶态及NH₄OAc提取态Zn、Cd的稳定效果明显优于磷矿粉,且在高污染土壤上效果更佳.田间试验结果显示,施用钙镁磷肥不仅可增加水稻产量,且可一定程度上降低水稻地上部的Zn、Cd吸收量.     

土壤pH对东南景天修复镉和锌污染土壤的影响

采用盆栽方法研究重金属(Zn、Cd)2种污染水平(T1处理:Zn 1200 mg·kg-1+Cd 20 mg·kg-1;T2处理:Zn 300 mg·kg-1+Cd 2.0 mg·kg-1)、4个p H水平(4.0、5.5、7.0和8.5)下,土壤Zn、Cd有效态的变化、东南景天吸收和积累特征以及植物对土壤中Zn、Cd的去除效果。结果表明,随着p H的降低,土壤Zn、Cd的有效态含量显著增大,东南景天吸收和积累土壤Zn、Cd的效率显著提高。T1处理时东南景天生物量在p H 5.5时最大,但与其他p H处理的差异并不显著;Zn、Cd在植物地上与地下部分的含量均在p H 4.0时最大。T2处理时东南景天在p H 4.0时体内重金属含量最高,但由于植物生长受到Al和Mn等元素的毒害,此时生物量最小。东南景天对土壤重金属去除率均在p H 5.5时最高,其中T1处理时Cd、Zn去除率分别为16%和1.33%,T2处理时分别为27%和1.09%;2种污染水平土壤重金属的去除率均在p H 8.5时最小。因此,适当降低土壤p H,可有效提高植物积累和去除土壤Zn、Cd的效率。本研究为进一步利用东南景天修复重金属污染土壤、提高植物修复效率提供了科学依据。     

麦季间作伴矿景天对不同土壤小麦-水稻生长及锌镉吸收性的影响

采用黑龙江黑土、河南潮土和浙江水稻土等我国粮食主产区典型土壤开展盆栽试验,研究小麦/伴矿景天间作、水稻轮作模式下Zn、Cd超积累植物伴矿景天对当季小麦和后茬水稻生长及重金属吸收性的影响,探索粮食作物主产区污染土壤边生产边修复技术的可行性.结果表明:麦季间作伴矿景天,土壤硝酸钠提取态Zn、Cd浓度较小麦单作处理显著提高,间作处理下水稻土、潮土与黑土的提取态Zn较单作处理分别提高55％、32％和110％,水稻土与黑土提取态Cd较单作分别提高38％和110％,潮土的提取态Cd与对照处理没有差异.间作处理水稻土、潮土和黑土上小麦地上部重金属浓度是单作处理的1.1～1.9倍.麦季间作伴矿景天对后茬水稻生长及其地上部重金属吸收性无显著影响,虽然后茬水稻糙米中Cd含量仍高于0.2 mg·kg-1的“食品中污染物限量”标准,但种植过伴矿景天处理的水稻糙米重金属与前季单作小麦处理相比呈下降趋势.表明通过伴矿景天/小麦-水稻的间作和轮作种植模式,可吸取修复污染土壤中有害重金属,降低后茬水稻的食物链风险.     

Involvement of histidine-rich domain of ZIP family transporter TjZNT1 in metal ion specificity.

The Zrt/Irt-like protein (ZIP) family generally contributes to metal homeostasis by regulating cation transport into the cytoplasm. Most ZIP members have a long variable loop between transmembrane domains III and IV, and these loops are predicted to be located in the cytoplasm. The loops contain a histidine-rich domain (HRD) postulated to serve as a metal ion binding site; however, its role has not yet been determined. We previously determined that deletion of the HRD did not affect the Ni tolerance ability of TjZNT1-a ZIP transporter that confers high Ni tolerance to yeast. In this study, we investigated the effect of HRD deletion on the ion transport ability of TjZNT1. The deletion of HRD increased the specificity for Zn2+, but not for Cd2+. In addition, we confirmed subcellular localizations of TjZNT1 and HRD-deleted mutants by green fluorescence protein (GFP)-fused proteins, indicating that the deletion of HRD did not affect the localization of TjZNT1. From these results, we propose that the HRD could be involved in the ion specificity of TjZNT1.     

Iron acquisition by phytosiderophores contributes to cadmium tolerance

Based on the ability of phytosiderophores to chelate other heavy metals besides iron (Fe), phytosiderophores were suggested to prevent graminaceous plants from cadmium (Cd) toxicity. To assess interactions between Cd and phytosiderophore-mediated Fe acquisition, maize (Zea mays) plants were grown hydroponically under limiting Fe supply. Exposure to Cd decreased uptake rates of 59Fe(III)-phytosiderophores and enhanced the expression of the Fe-phytosiderophore transporter gene ZmYS1 in roots as well as the release of the phytosiderophore 2'-deoxymugineic acid (DMA) from roots under Fe deficiency. However, DMA hardly mobilized Cd from soil or from a Cd-loaded resin in comparison to the synthetic chelators diaminetriaminepentaacetic acid and HEDTA. While nano-electrospray-high resolution mass spectrometry revealed the formation of an intact Cd(II)-DMA complex in aqueous solutions, competition studies with Fe(III) and zinc(II) showed that the formed Cd(II)-DMA complex was weak. Unlike HEDTA, DMA did not protect yeast (Saccharomyces cerevisiae) cells from Cd toxicity but improved yeast growth in the presence of Cd when yeast cells expressed ZmYS1. When supplied with Fe-DMA as a Fe source, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing a cauliflower mosaic virus 35S-ZmYS1 gene construct showed less growth depression than wild-type plants in response to Cd. These results indicate that inhibition of ZmYS1-mediated Fe-DMA transport by Cd is not related to Cd-DMA complex formation and that Cd-induced phytosiderophore release cannot protect maize plants from Cd toxicity. Instead, phytosiderophore-mediated Fe acquisition can improve Fe uptake in the presence of Cd and thereby provides an advantage under Cd stress relative to Fe acquisition via ferrous Fe.     

A cloned prokaryotic Cd2+ P-type ATPase increases yeast sensitivity to Cd2+

CadA, the P1-type ATPase involved in Listeria monocytogenes resistance to Cd(2+), was expressed in Saccharomyces cerevisiae and did just the opposite to what was expected, as it strikingly decreased the Cd(2+) tolerance of these cells. Yeast cells expressing the non-functional mutant Asp(398)Ala could grow on selective medium containing up to 100 microM Cd(2+), whereas those expressing the functional protein could not grow in the presence of 1 microM Cd(2+). The CadA-GFP fusion protein was localized in the endoplasmic reticulum membrane, suggesting that yeast hyper-sensitivity was due to Cd(2+) accumulation in the reticulum lumen. CadA is also known to transport Zn(2+), but Zn(2+) did not protect the cells against Cd(2+) poisoning. In the presence of 10 microM Cd(2+), transformed yeasts survived by rapid loss of their expression vector.     

Role of calcium channels in cadmium-induced disruption of cortisol synthesis in rainbow trout (Oncorhynchus mykiss)

The mechanisms of toxicity of cadmium (Cd(2+)) in adrenal steroidogenesis were investigated in vitro in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicity of Cd(2+) was increased in absence of extracellular Ca(2+), but was prevented in Ca(2+)-supplemented medium. Pretreatment of cells with BAY K8644 (BAY), an agonist of voltage-dependent calcium channels, increased the Cd(2+)-mediated inhibition of ACTH-stimulated secretion but not pregnenolone (PREG)-stimulated secretion. Nicardipine, an antagonist of voltage-dependent calcium channels, also increased the inhibition of adrenocorticotropic hormone (ACTH)-stimulated secretion by Cd(2+). These results suggest that opening of voltage-dependent calcium channels with BAY may allow Cd(2+) entry at the same time as calcium, thus increasing toxicity of Cd(2+), however voltage-dependent calcium channels may not be the only way of entry into adrenocortical cells. The influx of Cd(2+), measured as intracellular Cd(2+) using Fluo-3 in PREG-stimulated adrenocortical cells, was significantly enhanced by the stimulation. These results suggest that the deleterious effect of Cd(2+) on cortisol steroidogenesis may be enhanced when the endocrine stress response is triggered.