超积累植物伴矿景天镉耐受基因SpMT2的分离及功能鉴定

超积累植物由于其对重金属具有地上部超积累以及超耐受等特性,不仅是研究植物离子转运及毒性耐受的理想模式,而且在植物修复的发展和应用中具有不可替代的作用。伴矿景天是近年在我国境内发现的一种景天科镉(Cd)/锌(Zn)超积累植物。为鉴定其富集和耐受Cd的关键基因,笔者构建了其酵母表达cDNA文库,利用酵母的遗传互补系统筛选到一个极大提高了酵母抗Cd能力的基因SpMT2。SpMT2属于富含半胱氨酸(Cys)的金属硫蛋白(Metallothionein)家族。亚细胞定位表明SpMT2表达于酵母细胞质中,并特异地提高酵母对Cd的抗性。进一步研究发现SpMT2的表达显著降低了酵母液泡中Cd含量,但酵母吸收的总Cd含量无显著变化。推测抗性增加是由于SpMT2在酵母细胞质中通过螯合Cd从而降低Cd对酵母的毒害。qRT-PCR分析表明SpMT2在伴矿景天的根和地上部都高丰度表达,且不受Cd诱导变化。鉴于SpMT2也定位于植物细胞质中,结合上述结果,推测SpMT2可能在伴矿景天细胞质中螯合Cd,在降低Cd毒害的同时可能还保持Cd在细胞质中的流动性,从而在Cd长途转运过程中也发挥重要作用。     

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。     

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,并对今后的研究方向提出了一些看法。     

植物螯合肽合成酶的研究进展

植物螯合肽（phytochelatins,PCs）在植物解除重金属的毒性方面具有重要作用,其结构为（γ-Glu—Cys）n-Gly（n=2—11）,它不是基因的编码产物,而是在植物螯合肽合成酶（phytochelatin synthase,PCS）的催化下以谷胱甘肽（glutathione,GSH）为底物合成的。PCS能够被金属离子激活,高度保守的N-端是催化结构域,而其C-端则是多变的。本文就PCS的结构,功能与催化机制以及PCS的最新研究进行了介绍。     

植物络合素和植物络合素合酶的研究

植物络合素（Phytochelatins,PCs)是由于重金属离子诱导而在植物体内合成的一类小分子多肽,其结构式为（γ-Glu-Cys)n-Gly,(n=2-11);PCs能够螯合重金属,从而起到对对重金属解毒的作用,PCs并非基因的直接产物,而是由植物络合素合酶（phytochelatin syn-thase,PCS),以GSH为底物催化合成的;植物络合素合酶基因的表达是组成型的,重金属离子能够活化PCS,诱导PCs的合成。1989年,人们首次报道得到了部分纯化的PCS,10年后,3个研究小组分别于1999年同时克隆和鉴定了编码PCS的基因,这些结果不仅对于研究PCs的合成途径和模型的建立及植物抗重金属机制的探讨有重要意义,而且在利用基因工程改良植物抗重金属能力和净化环境污染方面有应用前景。     

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。     

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.     

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。     

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。     

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.     

Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescens (J. & C. Presl)

Thlaspi caerulescens (J. & C. Presl, "Prayon") is a heavy-metal hyperaccumulator that accumulates Zn and Cd to high concentrations (40,000 and 4,000 mg kg DW-1 respectively) without phytotoxicity. The mechanism of Cd tolerance has not been characterized but reportedly involves vacuolar sequestration. The role of phytochelatins (PCs) in metal tolerance in T. caerulescens and the related non-accumulator T. arvense was examined. Although PCs were produced by both species in response to Cd, these peptides do not appear to be involved in metal tolerance in the hyperaccumulator. Leaf and root PC levels for both species showed a similar positive correlation with tissue Cd, but total PC levels in the hyperaccumulator were generally lower, despite correspondingly higher metal concentrations. The lack of a role for PCs in the hyperaccumulator's response to metal stress suggests that other mechanisms are responsible Cd tolerance. The lower level of leaf PCs in T. caerulescens also implies that Cd in the shoot is sequestered in a compartment or form that does not elicit a PC response.     

Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non‐resistant plants

Phytochelatin (PC) synthesis is considered necessary for Cd tolerance in non‐resistant plants, but roles for PCs in hyper‐accumulating species are currently unknown. In the present study, the relationship between PC synthesis and Cd accumulation was investigated in the Cd hyperaccumulator Sedum alfredii Hance. PCs were most abundant in leaves followed by stems, but hardly detected by the reversed‐phase high‐performance liquid chromatography (HPLC) in roots. Both PC synthesis and Cd accumulation were time‐dependent and a linear correlation between the two was established with about 1:15 PCs : Cd stoichiometry in leaves. PCs were found in the elution fractions, which were responsible for Cd peaks in the anion exchange chromatograph assay. About 5% of the total Cd was detected in these elution fractions as PCs were found. Most Cd was observed in the cell wall and intercellular space of leaf vascular cells. These results suggest that PCs do not detoxify Cd in roots of S. alfredii. However, like in non‐resistant plants, PCs might act as the major intracellular Cd detoxification mechanism in shoots of S. alfredii.     

Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii

Sedum alfredii has been reported to be a cadmium (Cd) hyperaccumulator. Phytochelatins (PCs) and other thiol (SH)-containing compounds have been proposed to play an important role in the detoxification and tolerance of some heavy metals, but it is not clear whether PCs are responsible for Cd hyperaccumulation and tolerance in S. alfredii. In this study, two geographically isolated populations of S. alfredii were studied: one population grew on an old Pb/Zn mine site, while the other on a non-mine site. The mine population of this species exhibited a stronger heavy metal tolerance than in the other population. Root-to-shoot transport of Cd was higher in population located at the mine site than at the non-mine site. Considerable amounts of Cd were accumulated in leaves and stems of mine plants, while most Cd was distributed in roots of non-mine plants. Non-protein SH in plant tissues of two populations were further investigated by a HPLC pre-column derivatization system. Upon exposure to Cd, no PCs were detected in all tissues of mine population, while an appreciable amount of glutathione (GSH) was observed in the descending order of stem>root>leaf. The concentrations of GSH consistently increased with the increase of exogenous Cd concentrations and time. On the contrary, Cd exposure strongly induced the production of PCs (mainly PC(2) and PC(3)) and GSH in plant tissues of non-mine population, and the concentrations of GSH showed an initial drop over the duration of 7-d exposure. The present results provided strong evidence that PCs are not involved in Cd transport, hyperaccumulation and tolerance in mine population of S. alfredii.     

植物螯合肽合酶基因AtPCS2的表达调控

植物螯合肽合酶（pcs）受重金属离子激活,并以还原型谷胱甘肽为底物合成植物螯合肽（PCs）,在植物和真菌的重金属解毒机制中起重要作用．拟南芥基因组中有两个编码PCS的基因AtPCS1和AtPCS2,但AtPCS1单基因功能缺失即可导致相应的突变体cad1—3对镉高度敏感,其体内也检测不到PCs;而体外表达分析表明,AtPCS2具有完全的PCs合酶活性,预示植物体内可能存在AtPCS2的负向调控机制．基于该推测,构建了CaMV35S启动子驱动的AtPCS2基因编码区与c—Myc抗原标签融合的过表达载体．结果表叽在cadl-3的MV35S／AtPCS2：cMyc的异位表达株系中,AtPCS2的mRNA和蛋白都保持较高的表达量．不仅如此,AtPCS2具有植物螯合肽合成能力,并完全互补了cad1-3突变体的镉敏感性状．AtPCS2和EYFP的融合蛋白在细胞质有明显表达,在细胞核也检测到一定信号．以上结果表明,AtPCS2在植物体内可能主要受转录水平调控,而且可能具有调节PCs合成以外的其他生化功能．     

Long-term field phytoextraction of zinc/cadmium contaminated soil by Sedum plumbizincicola under different agronomic strategies

In two long-term field experiments the zinc (Zn)/cadmium (Cd) hyperaccumulator Sedum plumbizincicola (S. plumbizincicola) was examined to optimize the phytoextraction of metal contaminated soil by two agronomic strategies of intercropping with maize (Zea mays) and plant densities. Soil total Zn and Cd concentrations decreased markedly after long-term phytoextraction. But shoot biomass and Cd and Zn concentrations showed no significant difference with increasing remediation time. In the intercropping experiment the phytoremediation efficiency in the treatment “S. plumbizincicola intercropped with maize” was higher than in S. plumbizincicola monocropping, and Cd concentrations of corn were below the maximum national limit. In the plant density experiment the phytoremediation efficiency increased with increasing plant density and 440,000 plants ha^?1 gave the maximum rate. These results indicated that S. plumbizincicola at an appropriate planting density and intercropped with maize can achieve high remediation efficiency to contaminated soil without affecting the cereal crop productivity. This cropping system combines adequate agricultural production with soil heavy metal phytoextraction.     

植物对重金属镉的耐受机制

镉离子（Cd^2＋）具有强植物毒性,抑制植物生长,甚至使植物死亡。由于长期的环境选择和适应进化,植物发展出耐受机制,可减轻或避免Cd^2＋的毒害。硫转运蛋白、硫还原相关酶类以及半胱氨酸、谷胱甘肽和植物螯合肽合成基因的表达受Cd^2＋调控。同时这些基因的过表达也能提高植物对Cd^2＋的耐性。植物抗氧化系统对Cd^2＋胁迫诱发的活性氧的清除作用,具转运Cd^2＋活性的质膜转运蛋白促进Cd^2＋经共质体途径向木质部运输、装载,而后随蒸腾流向地上部迁移,具转运Cd^2＋活性的液泡膜转运蛋白促进Cd^2＋进入液泡的隔离作用,都在植物对Cd^2＋的耐性中起作用。     

Antioxidative defense and proline/phytochelatin accumulation in a newly discovered Cd-hyperaccumulator, Solanum nigrum L.

Changes in the activity of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and the contents of malondialdehyde (MDA), chlorophyll, free proline and phytochelatins (PCs) in Solanum nigrum, the newly discovered Cd-hyperaccumulator were examined and compared with a non-hyperaccumulator Solanum melongena. It was indicated that leaf SOD and POD activity of S. nigrum was significantly higher than that of S. melongena. The Cd treatments significantly increased root SOD activity, leaf POD activity, and CAT activity and free proline content in the leaves and roots of S. nigrum. On the contrary, the Cd treatments decreased SOD activity, and did not change CAT activity in the leaves and roots of S. melongena. Moreover, there were no significant differences in free proline levels in the roots of S. melongena. These results validated that S. nigrum had a greater capacity than S. melongena to adapt to oxidative stress caused by Cd and free proline accumulation might be responsible for the tolerance of S. nigrum to Cd. Treated with 10 μg Cd g⁻¹, growth of S. nigrum and its contents of chlorophyll and MDA were basically unaffected. In contrast, there were a decrease in the growth and chlorophyll content, and an increase in MDA in the roots of S. melongena. Although lipid peroxidation was promoted in both the hyperaccumulator and non-hyperaccumulator by high Cd stress, the greater increase took place in the tissues of S. melongena. The PCs level in roots of S. nigrum was significantly lower than that of S. melongena. On the contrary, the content of leaf PCs was much higher in S. nigrum than that in S. melongena. These results further suggested that antioxidative defense in the Cd-hyperaccumulator might play an important role in Cd tolerance, and PCs synthesis is not the primary reason for Cd tolerance although PCs in S. nigrum increased significantly by Cd.