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ABSTRACT: Solanum nigrum L. is considered to be a potential plant for restoring Cd-contaminated soils. Si could enhance plants tolerance to heavy metal; however, the mechanism of Si-mediated alleviation of Cd toxicity in S. nigrum was not clear. Three-week-old S. nigrum seedlings were grown in Hoagland solution containing 0 or 100 μM Cd with or without 1 mM Si for 4 days. The results showed that the Cd concentration both in roots and shoots of Si-supplied plant was significantly reduced, especially in expanding and old leaves. The relative proportion of ethanol-extractable Cd, water-extractable Cd and NaCl-extractable Cd in roots was increased by adding Si, while the root-to-shoot Cd translocation was not decreased. Furthermore, in comparison with single Cd treatment, supplying Si could reduce H2O2 accumulation and cell death in roots, and the electrolyte leakage and H2O2 concentration in functional leaves. Moreover, the activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11) in functional leaves was markedly increased by Cd exposure, while the antioxidative enzyme activities in Cd plus Si treatment seedlings were significantly lower than that in Cd treatment alone, this decrease might be attributed to the reduction of Cd concentration and Cd-induced oxidative damages. These results demonstrate that Si-enhanced Cd tolerance in S. nigrum is mainly due to the decrease of Cd uptake in roots and Cd distribution in expanding and old leaves, as well as lowering oxidative stress induced by Cd in plants.
Plant Physiology and Biochemistry 04/2013; 68C:1-7. · 2.84 Impact Factor
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ABSTRACT: An iterative combinatorial mutagenesis (ICM) strategy was used to engineer deacetoxycephalosporin C synthase of Streptomyces clavuligerus (scDAOCS) for improved activity toward penicillin G. Seven mutational sites were repeatedly combined onto a starter mutant (C155Y Y184H V275I C281Y) of scDAOCS. Eleven improved combinatorial mutants were identified from 24 mutants in four rounds of ICM.
Applied and environmental microbiology 08/2012; 78(21):7809-12. · 3.69 Impact Factor
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ABSTRACT: JadH is a bifunctional hydoxylase/dehydrase involved in jadomycin biosynthesis; it catalyzes a post-PKS modification reaction to convert 2,3-dehydro-UWM6 to dehydrorabelomycin. To identify the key residues involved in substrate-binding and catalysis, structural modeling and multiple sequence alignments of JadH homologs were performed to predict nine residues at the proximity of substrate. Site-directed mutagenesis of the corresponding residues and in vitro evaluation of the activities of the mutant enzymes, indicate these mutations severely reduced JadH activity. Our results indicate these residues are specifically involved in substrate-binding or catalysis in JadH.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2012; 28(8):950-8.
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ABSTRACT: Two component system is a signal transduction system. It typically consists of a sensor histitine kinase and a cognate response regulator (RR) component. The activity of RR is regulated by a phosphorylation dependent mechanism. In recent years, the existence of atypical response regulators (ARRs), which rely on a phosphorylation independent mechanism to regulate their activity, have been recognized. ARRs are involved in the regulation of bacterial growth and development, antibiotic biosynthesis, iron transport, among others. Here we review the recent advances in the understanding of the structure and function of atypical response regulators, by using JadR1, a regulator in jadomycin biosynthesis in Streptomyces, as an example to elucidate the novel mechanism used by ARR to fine-tune its activity.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 05/2012; 28(5):531-9.
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ABSTRACT: To explore the regulation of expansin expression under heavy metal stresses, a novel expansin gene, BjEXPA1, and its upstream region were obtained from Indian mustard (Brassica juncea L.). BjEXPA1 encodes a cell wall protein of the α-subgroup of the expansin family. The expression of BjEXPA1 is regulated by exogenous phytohormones, NaCl, and heavy metals. Sequence analysis of its upstream region revealed the presence
of several putative cis-acting elements, including phytohormone response, abiotic stress response, and tissue-specific elements. GUS activity under
the control of the BjEXPA1 promoter allowed observation of BjEXPA1 spatial and temporal expression patterns. The reporter construct indicated that BjEXPA1 is induced by exogenous gibberellin and auxin, and inhibited by abscisic acid, CdCl2 and PEG6000. Transgenic tobacco seedlings overexpressing BjEXPA1 showed an altered phenotype and enhanced sensitivity to cadmium. Higher H2O2 accumulation and extensive plasma membrane damage were detected in the leaves of transgenic seedlings when exposed to Cd,
compared with wild-type plants. Our findings support the conclusion that overexpression of BjEXPA1 in tobacco renders plants susceptible to Cd stress, probably due to disruption of the growth process and/or the breakdown
of cell walls.
KeywordsExpansin–Heavy metal–Promoter–Growth phenotype
Plant Growth Regulation 04/2012; 64(1):39-51. · 1.60 Impact Factor
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ABSTRACT: Dehydrins are capable of conferring abiotic stress tolerance in plants. Few dehydrins have been characterized at the molecular
and transgenic level in Brassica juncea. In the present study, two SK2-type B.juncea dehydrin genes, BjDHN2 and BjDHN3, showed improved stress tolerance to salt and freezing stress in transgenic yeast. Semiquantitative reverse transcription
polymerase chain reaction (RT-PCR) revealed that BjDHN2 and BjDHN3 were up-regulated by low temperature, drought, and salt stress, as well as exogenous abscisic acid (ABA) in B. juncea. Transgenic studies also showed that BjDHN2 and BjDHN3 increased tolerance to cold and salt stress in tobacco. These results indicate that the BjDHN2 and BjDHN3 genes exert a protective role on membranes and play a role in abiotic stresses.
Molecular Breeding 04/2012; 21(4):431-438. · 2.85 Impact Factor
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ABSTRACT: The present study aims to elucidate the role of antioxidative enzyme in the adaptive responses of metal-accumulators (Thlaspi caerulescens and Brassica juncea) and non-accumulator plant (Nicotiana tabacum) to Cadmium stress. When seedlings of plants were grown in hydroponic condition for a period of 4days in the presence of
200 or 400μM CdCl2, photosynthetic rate, transpiration rate and stomatal conductance in metal-accumulators decreased more slowly than that in
tobacco. MDA content and electrolyte leakage increased with elevated Cd concentration and exposure time in all plant species,
while the oxidative damage in tobacco was more serious than that in metal-accumulators. The activities of SOD and CAT in metal-accumulators
were significantly higher than that in tobacco under normal condition, whereas there was no significant difference in the
activity of POD between Indian mustard and tobacco. The activities of antioxidative enzymes increased rapidly in metal-accumulators
in response to the Cd treatments, especially SOD and CAT. In tobacco, CAT activity declined rapidly by exposure to the Cd
treatment, though the activity of SOD and POD was enhanced, indicating that the antioxidative enzymes in tobacco could not
fully scavenge ROS generated by Cd toxicity. These results collectively indicate that the enzymatic antioxidation capacity
is one of the important mechanisms responsible for metal tolerance in metal-accumulator plant species.
Plant and Soil 04/2012; 310(1):137-149. · 2.73 Impact Factor
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ABSTRACT: Cation transporters play important roles in modulating the concentration of intracellular metal ions. The vacuole is an important storage organelle for many ions. Cation (Ca+)/H+ antiporters (CAXs) located at vacuolar membrane are mainly involved in the Ca2+ flux into the vacuole, and appear to be capable of transporting various divalent cations to some degree. Several CAX genes have been isolated and characterized from various plants in recent years. Four domains of plant CAXs have been identified: NRR regulates Ca2+ transport by a mechanism of N-terminal autoinhibition; Ca domain and C domain confer Ca2+ and Mn2+ specificity among CAX transporters, respectively; D domain plays a part in the regulation of cytosolic pH. AtCAXs identified in Arabidopsis thaliana are involved in the growth, development and stress adaption of plant. AtCAX3 is the mainly Ca2+/H+ transporter in response to salt stress; AtCAX2 and AtCAX4 participate in transportation and detoxicification of heavy metal ions (Cd2+, Zn2+, and Mn2+) in cells under heavy metal stress, and impact root/shoot Cd partitioning in plant. These suggest that CAX genes may be useful for nutritional enhancement of plants, and for increasing phytoremediation potential. Here, the classification, structure and function of CAXs in plants are reviewed.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 04/2011; 27(4):546-60.
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ABSTRACT: The regulation of the heavy-metal accumulation in vivo for plant survival is very complex. The metal cation transporter plays key roles in the metabolic process. P(1B)-ATPases are the only subgroup of P-ATPases that contribute to heavy metal homeostasis presented in most organisms. Arabidopsis thaliana contains eight genes encoding P(1B)-ATPases. The current reports show that the functions of P(1B)-ATPases are involved in maintaining metal homeostasis, transporting and detoxification in plants. P(1B)-ATPases not only mediated metal ion mobilization and uptake in roots, but also contribute to the metal transport, storage and tolerance in shoots, especially in heavy metal hyperaccumulators. In this paper, we reviewed and discussed the evolution, classification, structure and function of P(1B)-ATPases in plants. HMAs-transgenic manipulation could be a feasible approach for phytoremediation and mineral nutrition fortification.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2010; 26(6):715-25.
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ABSTRACT: Catalase (CAT), an important enzyme of antioxidant system, was investigated the role in preventing the plant from Cd-induced oxidative stress caused by reactive oxygen species. A CAT gene from Brassica juncea was cloned and up-regulated in response to Cd/Zn. The CAT cDNA (BjCAT3) under the control of CaMV35S promoter was introduced into tobacco via Agrobacterium-mediated transformation. Northern blot analysis verified the BjCAT3 was expressed at high level in different transgenic lines. In morphological observation, we found that seedlings from transgenic tobacco plants grew better and showed longer root length in the presence of Cd versus wild-type (WT) seedlings. Under 100 microM Cd stress, WT plants became chlorotic and almost dead while transgenic tobacco plants still remained green and phenotypically normal. The CAT activity of transgenic T(1) generations was approximately two-fold higher than that of WT plants. In WT, endogenous CAT activity is rapidly reduced as a result of 200 microM CdCl2 exposure. Compared with WT plants, lower level of Cd-induced H2O2 accumulation and cell death were detected in roots of transgenic plants with high level of CAT activity. All our findings strongly support that overexpressing BjCAT3 in tobacco could enhance the tolerance under Cd stress.
Chemosphere 08/2009; 76(5):623-30. · 3.21 Impact Factor
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ABSTRACT: 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 Deltazrc1Deltacot1 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.
Plant Cell Reports 07/2009; 28(8):1235-42. · 2.27 Impact Factor
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ABSTRACT: Phytohormone auxins play important roles in plant growth and development. The primary auxin-response genes can be classified into three major groups: Aux/IAAs, SAURs and GH3s. Significant progress has been made in understanding these gene families by approaches of the functional genomics, molecular genetics and molecular biology. In this review, we focused on the structures, functions and models of the expressional regulation of plant GH3 genes. The interactions in the signal transduction pathways between auxins and other signals mediated by the GH3 genes, the relationship between the GH3 genes and the stress adaptation responses of plants are emphasized.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2008; 24(11):1860-6.
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ABSTRACT: An aquaporin cDNA BjPIP1 isolated from heavy-metal accumulator Indian mustard (Brassica juncea L.) encodes a 286-residue protein. The deduced amino acid sequence of BjPIP1 with six putative transmembrane domains showed highest identity (85-99%) to PIP1 subfamily members. Semi-quantitative RT-PCR analysis revealed that BjPIP1 transcripts were more abundantly expressed in roots compared to aerial parts of Indian mustard. However, the expression of BjPIP1 in leaves was up-regulated by drought, salt, low temperature, and heavy metal stress, suggesting that BjPIP1 was involved in resistance to abiotic stresses. BjPIP1 under the control of 35S promoter was introduced into tobacco mediated with Agrobacterium tumefaciens, the transgenic tobacco exhibited a lower water loss rate, a decreased transpiration rate, and stomatal conductance compared to the wild-type plants under osmotic stress, indicating that BjPIP1 might enhance plant drought resistance by decreasing transpiration via reducing stomatal conductance. Furthermore, overexpression of BjPIP1 in tobacco enhanced Cd resistance of root growth, and lowered transpiration rate and stomatal conductance upon Cd exposure, suggesting that BjPIP1 might increase heavy-metal resistance by maintaining reasonable water status in tobacco. Moreover, the BjPIP1-overexpressing plants showed higher activities of antioxidative enzymes, and lower level of electrolyte leakage and malondialdehyde content under Cd stress, indicating BjPIP1 might enhance the antioxidative activity and membrane integrity in transgenic plants. Taken together, these results suggested that BjPIP1 might improve plant heavy-metal resistance through alleviating water deficit and oxidative damage induced by metal ions.
Molecular Biotechnology 08/2008; 40(3):280-92. · 2.17 Impact Factor
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ABSTRACT: The NRAMP gene family encodes integral membrane protein and mediates the transport of Fe, however, its function in transport of toxic metal ions is not very clear in plants. TcNRAMP3 was isolated from Thlaspi caerulescens, and encoded a metal transporter member of the NRAMP family. TcNRAMP3 was predominantly expressed in roots of T. caerulescens by semi-quantitative RT-PCR. The expression of TcNRAMP3 was induced by iron starvation and by the heavy metals Cd and Ni in roots. TcNRAMP3 was able to rescue growth of an iron uptake fet3fet4 mutant yeast strain, suggesting a possible role in iron transport. Expression of TcNRAMP3 in yeast increased Cd sensitivity and Cd content, while it enhanced the Ni resistance and reduced Ni accumulation, indicating that TcNRAMP3 could accumulate Cd and exclude Ni in yeast. Furthermore, overexpression of TcNRAMP3 in tobacco resulted in slight Cd sensitivity of root growth and did not influence Ni resistance. These results suggested that TcNRAMP3 played a role in metal cation homeostasis in plant.
Molecular Biotechnology 08/2008; 41(1):15-21. · 2.17 Impact Factor
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ABSTRACT: A novel member of the WRKY gene family, designated TcWRKY53, was isolated from a cadmium (Cd)-treated Thlaspi caerulescens cDNA library by differential screening. WRKY proteins specifically bind to W-boxes, which are found in the promoters of many genes involved in defense and response to environmental stress. TcWRKY53 contains a 975-bp open reading frame encoding a putative protein of 324 amino acids. Homology searches showed that TcWRKY53 resembles similar WRKY domain-containing proteins from rice, parsley and tobacco, especially AtWRKY53 from Arabidopsis thaliana. Semi-quantitative RT-PCR showed that the expression of TcWRKY53 was strongly induced by various environmental stresses, including an excess of NaCl, drought, cold and the signal molecule salicylic acid (SA). The expression of TcWRKY53 in response to NaCl, drought and cold suggested a possible role of TcWRKY53 in abiotic stress response. However, physiological tests indicated that the expression of TcWRKY53 in tobaccos decreases tolerance to sorbitol during seedling root development. This was consistent with PEG6000 treatment of tobacco seedlings, and together these results indicate a negative modulation of TcWRKY53 in response to osmotic stress. Furthermore, two ethylene responsive factor (ERF) family genes, NtERF5 and NtEREBP-1, were negatively induced in TcWRKY53-overexpressing transgenic plants. In contrast, a LEA family gene, NtLEA5, showed no change, suggesting that TcWRKY53 might regulate the plant osmotic stress response by interacting with an ERF-type transcription factor rather than by regulating function genes directly.
Plant Cell Reports 05/2008; 27(4):795-803. · 2.27 Impact Factor
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ABSTRACT: Micropropagation system of Malus zumi was optimized by studying the influence of plant growth regulators and culture conditions. The axillary buds were used for
mutiplication of in vitro shoot culture on agar Murashige and Skoog (1962) (MS) medium with combination of 1mgl−1 BAP, 0.5mgl−1 NAA or 0.5mgl−1 IAA or 0.5mgl−1 IBA under 16h photoperiod. The shoot growth in culture was not significantly affected within a broad range (5.0–7.0) of
initial medium pH. The highest shoot (13) was obtained on medium containing 1.0mgl−1 BAP and 0.5mgl−1 IAA. Well-developed shoots, 35–50mm in length, were successfully rooted ex vitro at 86.3% by a 2-h-treatment with aqueous
solution containing MS salts and 100mgl−1 IBA prior to their planting in growing substrate composed of soil and vermiculite (1:1 v/v). The survival rate of transplantation
reached 88.0% when transferred to field condition.
Acta Physiologiae Plantarum 12/2007; 30(1):129-132. · 1.64 Impact Factor
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ABSTRACT: The bean (Phaseolus vulgaris) stress-related gene number 2 (PvSR2) gene responds to heavy metals but not to other forms of environmental stresses. To elucidate its heavy metal-regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region (-1623/+48) of PvSR2. Deletions from the 5' end revealed that a sequence between -222 and -147 relative to the transcriptional start site was sufficient for heavy metal-specific induction of the promoter region of PvSR2. Detailed analysis of this 76-bp fragment indicated that heavy metal-responsive elements were localized in two regions (-222/-188 and -187/-147), each of which could separately confer heavy metal-responsive expression on the beta-glucuronidase gene in the context of a minimal cauliflower mosaic virus 35S promoter. Region I (-222/-188) contains a motif (metal-regulatory element-like sequence) similar to the consensus metal-regulatory element of the animal metallothionein gene, and mutation of this motif eliminated the heavy metal-inducible function of region I. Region II (-187/-147) had no similarity to previously identified cis-acting elements involved in heavy metal induction, suggesting the presence of a novel heavy metal-responsive element. Transformed tobacco (Nicotiana tabacum) seedlings expressing beta-glucuronidase under control of the PvSR2 promoter region (-687/+48) showed heavy metal-specific responsive activity that depended on the type and concentration of the heavy metal and the type of organ. These findings further our understanding of the regulation of PvSR2 expression and provide a new heavy-metal-inducible promoter system in transgenic plants.
Plant physiology 02/2007; 143(1):50-9. · 6.53 Impact Factor
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ABSTRACT: A heavy metal responsive gene PvSR3 (GenBank accession number U54703) encoding an acid dehydrin was isolated from a mercuric chloride-treated bean (Phaseolus vulgaris L.) leaf cDNA library by differential screening using cDNAs derived from treated and untreated plants. The PvSR3 cDNA is 981-bp long and has a 606-bp open-reading frame with a 202-residue-deduced amino acid sequence. The PvSR3 sequence contains two conserved repeats of the characteristic lysine-rich K segment (EKKGIMDKIKEKLPG) preceded by an 8-serine residue stretch, whereas the Y segment (DEYGNP) conserved motif is absent. The deduced protein has a calculated molecular weight of 23 kDa and an isoelectric point of 5.2. Sequence similarity and comparative analysis showed that PvSR3 shares 70 and 73% similarity with the dehydrin of poplar and pepper, respectively. Southern hybridizations indicated that PvSR3 was a low copy-number gene. Northern blot analysis revealed that PvSR3 mRNA was weakly detected in seedling leaves. However, the gene expression was strongly stimulated by heavy metals, such as mercury, cadmium, arsenic, and copper, whereas virus infection and salt had little effect on it. In contrast, PvSR3 was not responsive to drought or abscisic acid (ABA), and was downregulated by UV radiation. Furthermore, PvSR3 was upregulated by the exogenous signaling molecules, including salicylic acid (SA) and hydrogen peroxide (H2O2). It is suggested that PvSR3 is extremely related to heavy metal stress, and might play an important role in metal detoxification and resistance to the damage caused by heavy metals.
Molecular Biotechnology 04/2006; 32(3):205-18. · 2.17 Impact Factor
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ABSTRACT: We have developed a PCR-enriched cDNA pooling method for enrichment of the complete 5′ ends of the target homology cDNA fragments
with just 1 conserved region needed. By using reverse transcription and a few rounds of PCR amplification, a full-length cDNA
population flanked by T7 and M13 primers was generated. Multiple complete 5′ ends of cDNA members of a gene family can subsequently
be enriched via PCR with M13 and degenerate primer mix priming at the 5′ end and the conserved region, and they migrate as
a single dense band when separated on an agarose gel. The enriched homologous cDNA fragments could be separated for subsequent
cloning and sequencing. The main advantages of our method are its speediness, simplicity, and cost-effectiveness. The method
has been successfully applied to the cloning of members of the cation-efflux family inBrassica juncea L. and the natural resistance-associated macrophage protein family inThlaspi caerulescens, which demonstrates that this novel approach permits rapid isolation of novel interspecific gene orthologues. It could also
be easily adapted to highly specific cloning of gene homologues identified in target genomes.
Plant Molecular Biology Reporter 08/2005; 23(3):219-226. · 2.45 Impact Factor
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ABSTRACT: 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 CdCl(2)), whereas transgenic plant showed a lower root Cd content than the control at higher external Cd concentration (0.1 mmol/L CdCl(2)). 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.
Science in China Series C Life Sciences 01/2004; 46(6):623-30. · 1.61 Impact Factor
