Cadmium resistance in transgenic tobacco plants enhanced by expressing bean heavy metal-responsive gene <Emphasis Type="Italic">PvSR2</Emphasis>

PvSR2 (Phaseolus vulgaris stress-related gene) has been cloned from French bean and shown to be expressed specifically upon heavy metal treatment. In order to investigate the role of PvSR2 in plant, PvSR2 gene under the control of cauliflower mosaic virus 35S promoter was introduced into tobacco mediated with Agrobacterium tumefaciens LBA4404. The regenerated plantlets were selected on medium with 100 mg/L kanamycin. PCR and Southern blot analysis showed PvSR2 gene was integrated in tobacco genome. Gus and Northern blot analysis indicated PvSR2 gene was expressed in transgenic seedling. The heavy metal resistance assay showed that the transgenic tobacco seedlings with the PvSR2 coding sequence exhibited higher tolerance to Cd compared with wild-type (WT) under Cd exposure. The Cd content accumulated in root between transgenic and WT seedlings had no obvious difference at lower Cd external concentration (0.05-0.075 mmol/L CdCl2), whereas transgenic plant showed a lower root Cd content than the control at higher external Cd concentration (0.1 mmol/L CdCl2). These results suggested that the expression of PvSR2 can enhance the Cd tolerance, and PvSR2 may be involved in Cd transportation and accumulation at the test concentration of 0.1 mmol/L Cd.     

小鼠金属硫蛋白突变体αα-cDNA在烟草中的表达及转基因烟草对Cd~(2+)抗性的提高

金属硫蛋白是一类小分子量的金属结合蛋白 ,有 β,α两个结构域 .用另一个 α结构域取代金属硫蛋白的 β结构域 ,得到突变体 αα.通过 PCR法在 αα- c DNA翻译起始密码子 ATG前加入植物偏爱的碱基组合 AACA,构建 Ca MV双 35S启动子驱动的 αα- c DNA植物表达载体 p GPTVd35S-αα,此载体以抗除草剂基因 ( bar)作为选择标记物 .用根癌农杆菌介导的叶盘法对栽培种烟草 NC89进行转化 .Southern和 Western印迹分析表明突变体αα- c DNA已整合进入烟草基因组并且可以正常表达 .Northern印迹结果显示突变体αα- c DNA在烟草根部的表达强于叶部 .烟草根、茎、叶Cd2 +含量分析表明 ,转基因植物与对照相比 ,Cd2 +更多地集中在根部 ,在一定程度上降低了叶片中的 Cd2 + 含量 .Cd2 + 抗性实验进一步证明突变体αα- c DNA的表达提高了转基因烟草对 Cd2 + 的抗性 :转基因烟草在 2 0 0μmol/L Cd Cl2 条件下生长正常 ,在 40 0μmol/Cd Cl2 培养基内也可以存活 .而 1 0 0μmol/L Cd Cl2 即成为对照烟草的致死剂量 .     

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl₂ over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.     

Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding gamma-glutamylcysteine synthetase (gamma-ECS), targeted to the plastids. The gamma-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, gamma-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, gamma-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the gamma-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of gamma-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of gamma-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

Inheritance and expression of the mouse metallothionein gene in tobacco: impact on cd tolerance and tissue cd distribution in seedlings

Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.     

Functional analyses of TaHMA2, a P1B‐type ATPase in wheat

Currently, there are few studies concerning the function of heavy metal ATPase 2 (HMA2), particularly in monocotyledons, and the potential application of this protein in biofortification and phytoremediation. Thus, we isolated and characterized the TaHMA2 gene from wheat (Triticum aestivum L.). Our results indicate that TaHMA2 is localized to the plasma membrane and stably expressed, except in the nodes, which showed relatively high expression. Zinc/cadmium (Zn/Cd) resistance was observed in TaHMA2‐transformed yeast. The over‐expression of TaHMA2 increased the elongation and decreased the seed‐setting rate in rice (Oryza sativa L.), but not Arabidopsis thaliana, tobacco (Nicotiana tabacum L.) or wheat. TaHMA2 over‐expression also improved root‐shoot Zn/Cd translocation, especially in rice. The seeds of transgenic rice and wheat, not tobacco, showed decreased Zn concentrations. The Zn concentration was decreased in all parts of the transgenic rice seeds, but was decreased only in the ventral endosperm of wheat, which showed an increased Zn concentration in the embryo and aleurone. The over‐expression of TaHMA2 improved plant tolerance under moderate Zn stress and Zn deficiency, but Zn and Cd resistance decreased under high levels of Zn and Cd stress, respectively. The Cd concentration in transgenic rice seedlings was dramatically increased under Zn deficiency. Thus, over‐expression of TaHMA2 showed a more obvious phenotype in monocotyledons than in dicotyledons. These findings provide important information for TaHMA2, and more efforts should be made in the future to characterize the reduced Zn concentration in TaHMA2 transgenic grains and the diversity of TaHMA2 substrate specificity.     

Transgenic Tobacco with αα Mutant Gene Has Higher Tolerance to Heavy Metals

Metallothionein (MT) has two domains, α and β domain. α domain preferred to bind Cd2+and Hg2+. Mouse metallothionein mutant αα has been constructed and expressed in E.coli, which has the same stability as the nature one but has stronger affinity to heavy metals. To testify the result in vivo, αα mutant gene was cloned into plant expression vector pE3 under the CaMV 35S promoter. A transgenic tobacco was obtained by using leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) to Agrobacterium-mediated ααgene transfer. Southern blotting analysis indicated that the αα mutant gene was indeed integrated into the tobacco genome; Western blot indicated that the αα mutant gene was expressed in transgenic tobacco. It was also demonstrated that the transgenic tobacco with αα mutant gene have a little higher tolerance to heavy metals than that with natural MT gene. Moreover, the transgenic tobacco can accumulate more Cd2+ in its roots than natural, so that, it can decrease the concentration of Cd2+ in its leaves.     

转彩色马铃薯StAN1基因的烟草幼苗耐旱性分析

该研究以转彩色马铃薯StAN1基因烟草为材料、野生型烟草(WT)为对照,测定分析转StAN1基因烟草在种子萌发期、幼苗期和苗期对干旱(甘露醇)处理的耐受情况,并对苗期旱热共同胁迫的耐受情况进行测定分析,以探讨彩色马铃薯StAN1基因的功能,为耐旱彩色马铃薯育种提供新路径。结果显示:(1)转StAN1基因烟草鉴定显示,阳性率为82.6%,且转基因烟草的叶片明显变紫,花青素含量极显著高于野生型烟草。(2)在培养基甘露醇浓度为150 mmol/L时,点播在培养基上的转基因烟草种子第5天时的萌发率达到了7%,是野生型烟草萌发率的2.3倍。(3)在甘露醇浓度为0和100 mmol/L的培养基上竖直培养时,转基因烟草的根长分别是野生型烟草的1.46和1.30倍,根长比野生型烟草显著增长。(4)在干旱胁迫下,转基因烟草幼苗叶片中的脯氨酸含量以及超氧化物歧化酶活性均显著高于野生型烟草,丙二醛含量均显著低于野生型烟草。(5)转基因烟草LEA基因和ERF基因在干旱和旱热处理中的相对表达量均高于野生型烟草。研究表明,StAN1基因在提高植物花青素含量的同时也提高了植物的耐旱性。     

磷和镉对根内球囊霉Glomus intraradices孢子萌发、菌丝生长和外生菌丝内聚磷酸累积的影响

丛枝菌根真菌(AMF)能够显著提高植物对重金属的抗性,菌丝内聚磷酸(Polyphosphate,PolyP)可能参与了这种抗性的形成。试验以Glomus intraradices孢子为试材,对灭菌条件进行了优化,并进一步研究了不同P和Cd2+水平对孢子萌发、菌丝生长、分支和外生菌丝中聚磷酸含量的影响。结果表明,孢子在1%氯胺T+0.02%链霉素+0.01%庆大霉素+1/100(V/V)吐温-20中灭菌5min的效果最好。孢子萌发率、菌丝分支和菌丝长度随着Cd2+浓度的增加不断降低;当Cd2+浓度达到0.1mmol/L时,孢子萌发率降低为0%,表明Glomus intraradices的孢子萌发对Cd2+的耐受极限为0.1mmol/L;1mmol/L的P促进菌丝分支增加,却降低了萌发率,但对菌丝生长没有影响;在培养23d以后,三者基本不再变化。外生菌丝内的聚磷酸含量随着P的升高而增加;在Cd2+胁迫作用下,聚磷酸的含量降低而菌丝密度随着聚磷酸的升高而升高,表明聚磷酸在减弱重金属毒性方面起了重要作用。     

Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding γ-glutamylcysteine synthetase (γ-ECS), targeted to the plastids. The γ-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, γ-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, γ-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the γ-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of γ-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of γ-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

Isolation of novel types of Arabidopsis mutants with altered reactions to cadmium: cadmium-gradient agar plates are an effective screen for the heavy metal-related mutants

We are interested in elucidating the molecular mechanisms underlying plant reactions to the toxic heavy metal cadmium (Cd). To this end, we devised a new screening strategy using agar plates with a gradient of Cd concentrations, termed Cd-gradient agar plates (CGAPs), to isolate Arabidopsis mutants that displayed altered reactions to the metal. Arabidopsis M₂ seeds, derived from ethyl methanesulfonate (EMS) treated seeds, were germinated on the CGAPs such that the primary root of each seedling elongated against increasing concentrations of Cd on the surface of the plate. Under these conditions, the lengths of the primary roots reliably demonstrated the degree of Cd tolerance of individual seedlings. The use of CGAPs also allowed close observation of the root reaction of each seedling to Cd without causing lethal damage. The screen identified three mutant lines, MRC-32, MRC-22 and MRC-26, which showed distinctly different characteristics. MRC-32 plants exhibited enhanced tolerance to Cd and contained Cd at higher concentrations than wild-type (WT) plants treated with the heavy metal. The whole root system of MRC-22 plants showed a Cd-phobic response. MRC-26 plants accumulated less Cd in their aboveground tissues than WT plants, suggesting that they were defective in transporting the heavy metal from roots to aboveground tissues. We also determined the likely chromosomal location of each mutation.     

菜豆泛肽基因在生物和非生物胁迫下的表达

差别筛选HgCl2胁迫处理的菜豆（Phaseolus vulgaris L.）幼苗叶片cDNA库,分离出1个重金属胁迫响应基因PvSR52克隆,其cDNA长度为281bp。cDNA和氨基酸序列同源性分析表明PvSR52编码一种多聚泛肽。Southern blot结果表明菜豆泛肽可能由少数基因编码。Northern blot分析表明多聚泛肽叶片中表达较少;重金属Hg、Cd和As等、过量的Zn和Cu及高温、病毒侵染和水杨酸等环境胁迫均能强烈地刺激其在叶片中的表达。推测泛肽水解系统在提高植物的抗塑性方面有重要作用。     

霞多丽苗木中镉的积累、亚细胞分布及化学存在形态

以一年生盆栽葡萄品种霞多丽(Vitis vinifera L.cv.Chardonnay)自根苗及SO4砧嫁接苗为试材,采用CdCl2和CaCl2根部灌入的方法,研究了镉在霞多丽植株内的亚细胞分布和存在形态,以及外源添加氯化钙对植株镉吸收的影响.结果表明:大部分镉积累在霞多丽自根苗及嫁接苗的地下部器官;4 mmol·L-1浓度的CdCl2处理下,镉在自根苗根及根颈部分的积累量占77.1％,在叶片中的积累量占1.4％,而嫁接苗中镉在嫁接口以下部分的积累量高达93.9％,叶片中的积累量仅占0.1％;5 mmol·L-1外源钙缓解了植株对镉的吸收积累,而10mmol ·L-1外源钙则显著增加了植株对镉的吸收积累.镉在根系和叶片中的亚细胞分布规律为细胞壁＞可溶性部分＞细胞器,且在细胞壁中积累50％以上;镉在根系中主要以氯化钠提取态存在,其次为乙酸提取态,去离子水提取态含量最少.随着镉处理浓度的增加,各提取态含量有所变化.     

菜豆病程相关蛋白基因在重金属胁迫下的表达分析

为探讨植物抗重金属的分子机理 ,差别筛选了 Hg Cl2 胁迫的菜豆 ( Phaseolus vulgaris L.)叶片 c DNA库 ,分离出一个重金属胁迫响应基因 Pv SR4克隆 .c DNA和氨基酸序列分析表明 Pv SR4编码一种细胞内病程相关蛋白 ,该蛋白具有 RNase活性 .Northern blot分析表明 Pv SR4基因在正常生长条件下的叶片中不表达 ,重金属 ( Hg、Cd、As、Zn和 Cu等 )和水杨酸能强烈地诱导其基因的表达 ,受伤也能促进该基因的转录 ,而热胁迫几乎没有调节作用 .推测 Pv SR4蛋白在诱导植物的抗逆性和抵抗重金属胁迫方面有重要作用     

The cation-efflux transporter BjCET2 mediates zinc and cadmium accumulation in <Emphasis Type="Italic">Brassica juncea</Emphasis> L. leaves

Brassica juncea L. is a Zn/Cd accumulator. To determine the physiological basis of its metal accumulation phenotype, the functional properties and role of the metal efflux transporter BjCET2 were investigated using transgenic technology. Heterologous expression of BjCET2 in the double mutant yeast strain Δzrc1Δcot1 enhanced the metal tolerance of the yeast strain and led to decrease in Zn or Cd accumulation. Detection of green fluorescence from green fluorescent protein (GFP) in the root tip of transgenic tobacco further revealed that BjCET2::GFP is localized at the plasma membrane. Semi-quantitative RT-PCR analysis showed that BjCET2 was most abundant in the root and was weakly expressed in the stem and leaves. The expression of BjCET2 was up-regulated by heavy metals. However, exposure to low temperature, salt and drought did not affect the expression of BjCET2. Overexpression of BjCET2 in transgenic B. juncea plants conferred heavy metal tolerance and increased Cd/Zn accumulation in the leaves. BjCET2-deficient B. juncea mediated by antisense RNA resulted in hypersensitivity to heavy metals and decreased Zn/Cd accumulation in the plants. These results suggest that the heavy metal efflux of BjCET2 plays important roles in the metal tolerance of B. juncea and in Zn/Cd accumulation in B. juncea.     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Expression of BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana seedlings

The protective function of a plant type-2 metallothionein was analysed after expression in Escherichia coli and in Arabidopsis thaliana seedlings. BjMT2 from Brassica juncea was expressed in E. coli as a TrxABjMT2 fusion protein. After affinity chromatography and cleavage from the TrxA domain, pure BjMT2 protein was obtained which strongly reacted with the thiol reagent monobromobimane. Escherichia coli cells expressing the TrxABjMT2 fusion were more tolerant to Cu2+ and Cd2+ exposure than control strains. Likewise, when BjMT2 cDNA was expressed in A. thaliana under the regulation of the 35S promoter, seedlings exhibited an increased tolerance against Cu2+ and Cd2+ based on shoot growth and chlorophyll content. Analysis of transiently transformed cells of A. thaliana and tobacco leaves by confocal laser scanning microscopy (CLSM) revealed exclusive cytosolic localization of a BjMT2::EGFP (enhanced green fluorescent protein) fusion protein in control and heavy metal-exposed plant cells. Remarkably, ectopic expression of BjMT2 reduced root growth in the absence of heavy metal exposure, whereas in the presence of 50 or 100 microM Cu2+ root growth in control and transgenic lines was identical. The results indicate that in A. thaliana, root and shoot development are differentially affected by ectopic expression of BjMT2.