Generation of Mercury-Hyperaccumulating Plants through Transgenic Expression of the Bacterial Mercury Membrane Transport Protein MerC

The merC gene from Acidithiobacillus ferrooxidans functions as a mercury uptake pump. MerC protein localizes in the cytoplasmic membrane of plant cells. When Arabidopsis thaliana and tobacco plants were transformed with the merC gene under the control of the Cauliflower mosaic virus 35S promoter, the resulting overexpression of merC rendered the host plants hypersensitive to Hg²⁺ and they accumulated approximately twice as much Hg²⁺ ion as the wild type plants. Thus, bacterial mercuric ion transporters such as MerC may be useful molecular tools for producing transgenic plants that hyperaccumulate Hg²⁺ ion.     

The effect of the heterologous expression of Phragmites australis γ‐glutamylcysteine synthetase on the Cd2+ accumulation of Agrostis palustris

Heavy metal pollution has become one of the most serious environmental problems today. To develop a more efficient plant to clean up heavy metal contaminated soils, a γ‐glutamylcysteine synthetase (GCS) cDNA, named PaGCS, was isolated by PCR from Phragmites australis. The PaGCS sequence was transformed via agroinfection into the heavy metal intolerant grass Agrostis palustris. Five confirmed transgenic A. palustris plants expressing PaGCS were compared with the wild‐type line for growth and Cd²⁺ accumulation, as well as for the expression of a number of phytochelatin synthesis and stress‐responsive enzymes when challenged with Cd²⁺ stress. GCS and phytochelatin synthase (PCS) were up‐regulated in the transgenic lines. All the transgenic lines accumulated more Cd²⁺ and phytochelatins (PCs) than the wild‐type line, and three of the five lines grew more effectively than the wild‐type after either five or 21 d of Cd²⁺ stress. Variation among the transgenics was observed for the distribution of Cd²⁺ in the root, shoot and leaf. The malondialdehyde content of all the transgenic lines was lower than that of the wild type under Cd²⁺ treatment, while the activity of both superoxide dismutase and peroxidase present in the transgenic lines increased markedly 24 h after Cd²⁺ stress, and then rapidly declined.     

Engineering expression of bacterial polyphosphate kinase in tobacco for mercury remediation

To develop the potential of plants to sequester and accumulate mercurials from the contaminated sites, we engineered a tobacco (Nicotiana tabacum) plant to express a bacterial ppk gene, encoding polyphosphate kinase (PPK), under control of a plant promoter. The designated plant expression plasmid pPKT116 that contains the entire coding region of ppk was used for Agrobacterium-mediated gene transfer into tobacco plants. A large number of independent transgenic tobacco plants were obtained, in some of which the ppk gene was stably integrated in the plant genome and substantially translated to the expected PPK protein in the transgenic tobacco. The presence of Hg²⁺ did not cause considerable morphological abnormalities in the transgenic tobacco, which grew, flowered, and set seed similarly to the wild-type tobacco on the medium containing normally toxic levels of Hg²⁺. The ppk-transgenic tobacco showed more resistance to Hg²⁺ and accumulated more mercury than its wild-type progenitors. These results suggest that ppk-specified polyphosphate has abilities to reduce mercury toxicity, probably via chelation mechanism, and also to accumulate mercury in the transgenic tobacco. Based on the results obtained in the present study, the expression of ppk gene in transgenic tobacco plants might provide a means for phytoremediation of mercury pollution.     

Stable Integration and Functional Expression of Flounder Growth Hormone Gene in Transformed Microalga, Chlorella ellipsoidea

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 μg of fGH protein expression per one liter culture containing 1 × 10⁸ cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding. Received March 26, 2001; accepted July 10, 2001.     

Expression of merA,trxA, amoA,and hao in continuously cultured Nitrosomonas europaea cells exposed to cadmium sulfate additions

The effects of CdSO₄ additions on the gene expressions of a mercury reductase, merA, an oxidative stress protein, trxA, the ammonia‐monooxygenase enzyme (AMO), amoA, and the hydroxylamine oxidoreductase enzyme (HAO), hao, were examined in continuously cultured N. europaea cells. The reactor was fed 50 mM NH₄+ and was operated for 78 days with a 6.9 days hydraulic retention time. Over this period, six successive batch additions of CdSO₄ were made with increasing maximum concentrations ranging from 1 to 60 µM Cd²⁺. The expression of merA was highly correlated with the level of Cd²⁺ within the reactor (Rs = 0.90) with significant up‐regulation measured at non‐inhibitory Cd²⁺ concentrations. Cd²⁺ appears to target AMO specifically at lower concentrations and caused oxidative stress at higher concentrations, as indicated by the SOURs (specific oxygen uptake rates) and the up‐regulation of trxA. Since Cd²⁺ inhibition is irreversible and amoA was up‐regulated in response to Cd²⁺ inhibition, it is hypothesized that de novo synthesis of the AMO enzyme occurred and was responsible for the observed recovery in activity. Continuously cultured N. europaea cells were more resistant to Cd²⁺ inhibition than previously examined batch cultured cells due to the presence of Mg²⁺ and Ca²⁺ in the growth media, suggesting that Cd²⁺ enters the cell through Mg²⁺ and Ca²⁺ import channels. The up‐regulation of merA during exposure to non‐inhibitory Cd²⁺ levels indicates that merA is an excellent early warning signal for Cd²⁺ inhibition. Biotechnol. Bioeng. 2009; 104: 1004–1011. © 2009 Wiley Periodicals, Inc.     

Prevention by Ce3+ of DNA destruction caused by Hg2+ in fish intestine

The interactions between Hg²⁺, Ce³⁺, and the mixuure of Ce³⁺ and Hg²⁺, and DNA from fish intestine in vitro were investigated by using absorption spectrum and fluorescence emission spectrum. The ultraviolet absorption spectra indicated that the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ to DNA generated an obviously hypochromic effect. Meanwhile, the peak of DNA at 205.2 nm blue-shifted and at 258.2 nm red-shifted. The size of the hypochromic effect and the peak shift of DNA by metal ion treatments was Hg²⁺>Hg²⁺+Ce³⁺>Ce³⁺. The fluorescence emission spectra showed that with the addition of Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ the emission peak at about 416.2 nm of DNA did not obviously change, but the intensity reduced gradually and the sequence was Hg²⁺>Hg²⁺+Ce²⁺>Ce³⁺. Hg²⁺, Ce³⁺, and the mixture of Ce³⁺ and Hg²⁺ had 1.12, 0.19, and 0.41 binding sites to DNA, respectively; the fluorescence quenching of DNA caused by the metal ions all attributed to static quenching. The binding constants (K _A ) of binding siees were 8.98×10⁴ L/mol and 1.02×10⁴ L/mol, 5.12×10⁴ L/mol and 1.10×10³ L/mol, 6.66×10⁴ L/mol and 2.36×10³ L/mol, respectively. The results showed that Ce³⁺ could relieve the destruction of Hg²⁺ on the DNA structure.     

Light-Induced Hg Volatilization and O2 Evolution in Chlorella and the Effect of DCMU and Methylamine

The rate of volatilization of Hg²⁺ as metallic Hg is accelerated by illumination of Chlorella cells. In the presence of the uncoupler methylamine the rate of volatilization in the light is greatly but transiently increased. DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl urea) prevented the light response. In the presence of Hg²⁺, O₂ evolution by the cells was not completely inhibited by DCMU. Hg²⁺ appears to prevent DCMU reaching its binding site. Light seems to increase the amount of or leakage from the cells of a metabolite capable of reducing Hg²⁺ to Hg°.     

Development of a transgenic tobacco plant for phytoremediation of methylmercury pollution

To develop the potential of plant for phytoremediation of methylmercury pollution, a genetically engineered tobacco plant that coexpresses organomercurial lyase (MerB) with the ppk-specified polyphosphate (polyP) and merT-encoding mercury transporter was constructed by integrating a bacterial merB gene into ppk/merT-transgenic tobacco. A large number of independent transgenic tobaccos was obtained, in some of which the merB gene was stably integrated in the plant genome and substantially translated to the expected MerB enzyme in the transgenic tobacco. The ppk/merT/merB-transgenic tobacco callus showed more resistance to methylmercury (CH₃Hg⁺) and accumulated more mercury from CH₃Hg⁺-containing medium than the ppk/merT-transgenic and wild-type progenitors. These results suggest that the MerB enzyme encoded by merB degraded the incorporated CH₃Hg⁺ to Hg²⁺, which then accumulated as a less toxic Hg-polyP complex in the tobacco cells. Phytoremediation of CH₃Hg⁺ and Hg²⁺ in the environment with this engineered ppk/merT/merB-transgenic plant, which prevents the release mercury vapor (Hg⁰) into the atmosphere in addition to generating potentially recyclable mercury-rich plant residues, is believed to be more acceptable to the public than other competing technologies, including phytovolatilization.     

A comparative study of antioxidative defense system in the copper and temperature acclimated strains of <Emphasis Type="Italic">Anabaena doliolum</Emphasis>

This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu²⁺ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu²⁺ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu²⁺ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu²⁺ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu²⁺ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu²⁺ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu²⁺ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu²⁺ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu²⁺ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu²⁺ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu²⁺ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu²⁺ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM).     

MARINICHLORELLA KAISTIAE GEN. ET SP. NOV. (TREBOUXIOPHYCEAE,CHLOROPHYTA) BASED ON POLYPHASIC TAXONOMY1

We describe three coccoid green algal strains belonging to a new genus and species, Marinichlorella kaistiae Z. Aslam, W. Shin, M. K. Kim, W.‐T. Im et S.‐T. Lee, in seawater samples from the South Sea of Korea. These strains were maintained at 25°C–30°C under a 12:12 light:dark (L:D) photoregime in an ASN‐III medium at a pH of 7.5. These strains were tolerant of high salinity (7.5% NaCl) (w/v) and temperature (40°C). Molecular phylogenetic analyses using 18S rRNA gene sequence data resolved these organisms to a clade separate from green coccoid algae with similar morphology. The DNA–DNA hybridization results demonstrated very low relatedness of these organisms to phylogenetically related species of the genera Chlorella and Parachlorella. The molar guanine + cytosine content (G + C mol%) of the genomic DNA of these organisms ranged from 64.7 to 69.1 mol%. Based on molecular phylogeny, DNA–DNA hybridization, and other morphological studies, we propose a new taxon, Marinichlorella kaistiae, to describe these strains and classify them in the family Chlorellaceae. The type strain is KAS007^T (= KCTC AG10303^T = IAM C‐620^T).     

Lhcb2 gene expression analysis in two ecotypes of Sedum alfredii subjected to Zn/Cd treatments with functional analysis of SaLhcb2 isolated from a Zn/Cd hyperaccumulator

The Lhcb2 gene from hyperaccumulator Sedum alfredii was up-regulated more than three-fold while the non-hyperaccumulator accumulated one or two-fold higher amount of the mRNA than control plants under different concentrations of Cd²⁺ for 24 h. Lhcb2 expression was up-regulated more than five-fold in a non-hyperaccumulator S. alfredii when exposed to 2 μM Cd²⁺ or 50 μM Zn²⁺ for 8 d and the hyperaccumulator had over two-fold more mRNA abundance than the control plants. Over-expression of SaLhcb2 increased the shoot biomass by 14–41% and the root biomass by 21–57% without Cd²⁺ treatment. Four transgenic tobacco lines (L5, L7, L10 and L11) possessed higher shoot biomass than WT plants with Cd²⁺. Four transgenic lines (L7, L8, L10 and L11) accumulated 6–35% higher Cd²⁺ amounts in shoots than the wild type plants.     

过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。     

Bacterial <Emphasis Type="Italic">mer</Emphasis> operon-mediated detoxification of mercurial compounds: a short review

Mercury pollution has emerged as a major problem in industrialized zones and presents a serious threat to environment and health of local communities. Effectiveness and wide distribution of mer operon by horizontal and vertical gene transfer in its various forms among large community of microbe reflect importance and compatibility of this mechanism in nature. This review specifically describes mer operon and its generic molecular mechanism with reference to the central role played by merA gene and its related gene products. The combinatorial action of merA and merB together maintains broad spectrum mercury detoxification system for substantial detoxification of mercurial compounds. Feasibility of mer operon to coexist with antibiotic resistance gene (amp ^r , kan ^r , tet ^r ) clusters enables extensive adaptation of bacterial species to adverse environment. Flexibility of the mer genes to exist as intricate part of chromosome, plasmids, transposons, and integrons enables high distribution of these genes in wider microbial gene pool. Unique ability of this system to manipulate oligodynamic property of mercurial compounds for volatilization of mercuric ions (Hg²⁺) makes it possible for a wide range of microbes to tolerate mercury-mediated toxicity.     

菘蓝IiEMF基因的克隆与功能分析

该研究以菘蓝(Isatis indigotica Fort.)转录组数据为基础,克隆得到菘蓝EMF基因的cDNA全长,命名为IiEMF。(1)序列分析表明,IiEMF基因开放阅读框长度为1896 bp,编码631个氨基酸。进化树分析表明,菘蓝IiEMF蛋白与甘蓝(Brassica oleracea)EMF蛋白亲缘关系最为接近。(2)实时定量PCR结果显示,IiEMF在菘蓝不同器官中均有表达,且在叶中表达量最高,果实中表达量最低;IiEMF基因在菘蓝抽薹开花过程中叶内的表达量呈先升后降的趋势,并于初花期表达量达到最高后逐渐降低回落;在花/果期IiEMF基因表达量较花蕾中明显降低。(3)成功构建了超表达载体pCAMBIA1300-EMF,经农杆菌介导侵染拟南芥,PCR鉴定表明,有7株为超表达转IiEMF基因植株。(4)表型观察发现,在长日照和短日照条件下,与野生型相比2个转IiEMF基因拟南芥株系的开花时间都明显较早(提前6~10 d),且转IiEMF基因株系的莲座叶数比野生型多10片以上,叶片也比野生型大而肥厚。(5)qRT-PCR检测结果显示,在拟南芥营养生长过程中,过表达IiEMF显著抑制了拟南芥AtAP1、AtCO和AtLFY的表达,而促进了AtFLC的表达;当拟南芥开花时,转基因株系中的AtAP1和AtFLC表达量均高于野生型,AtCO和AtLFY的表达量显著低于野生型。研究表明,过量表达IiEMF基因能够促使拟南芥提前开花,且IiEMF可能是通过影响多种开花途径来共同调节促进拟南芥的早花。     

Genetic transformation of Brassica campestris var. rapa protoplasts with an engineered cauliflower mosaic virus genome

Summary A hybrid Cauliflower Mosaic Virus (CaMV) genome containing a selectable marker gene was constructed by replacing the gene VI coding region with the aminoglycoside (neomycin) phosphotransferase type II [APH(3)II] gene from Tn5. This modified viral genome was tested for its infectivity both in planta and in a protoplast transformation system of Brassica campestris var. rapa. Stable, genetically transformed cell lines of B. campestris var. rapa were obtained after transformation. DNA of the hybrid CaMV genome was found to be integrated into high molecular weight plant genomic DNA. Transformation was achieved only when the hybrid genome was supplied together with wild type viral DNA. A possible complementation of the modified CaMV genome with the wild type viral DNA as a helper molecule in planta and in the protoplast system is discussed.     

Transformation by Agrobacterium rhizogenes and regeneration of transgenic shoots of the wild soybean Glycine argyrea

Glycine argyrea accession G1420 was evaluated for its response to inoculation with Agrobacterium rhizogenes strains LBA9402 and A4T, carrying wild type Ri plasmids, and by strains R1601 and A4TIII with engineered plasmids. Hypocotyls from young seedlings were the most responsive in producing roots at inoculation sites. Root production was also dependent on bacterial concentration. Excised, cultured roots produced green nodular callus which regenerated shoots on SC2 medium containing 1.1 mg l^–1 6-benzylaminopurine and 0.005 mg l^–1 indole-3-butyric acid. The transformed nature of the roots and of callus regenerating shoots was confirmed by the presence of opines and by dot blot analysis for Ri TL-DNA. Tissues regenerated from roots transformed by A. rhizogenes strains R1601 and A4TIII exhibited NPTII enzyme activity, confirming the stable integration and expression of the chimaeric kanamycin resistance gene in transgenic tissues.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - NPTII neomycin phosphotransferase II - SDS sodium dodecyl sulphate