Arsenic resistance in Pteris vittata L.: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli
| Autor: | Lena Q. Ma, Shan Wu, Mrittunjai Srivastava, Jean Rivoal, Bala Rathinasabapathi, Sabarinath Sundaram |
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| Rok vydání: | 2006 |
| Předmět: |
DNA
Complementary Molecular Sequence Data Drug Resistance chemistry.chemical_element Plant Science medicine.disease_cause Gene Expression Regulation Enzymologic Triosephosphate isomerase chemistry.chemical_compound Cytosol parasitic diseases Escherichia coli Genetics medicine Amino Acid Sequence Cloning Molecular Phylogeny Arsenic Plant Proteins Arsenite Dose-Response Relationship Drug Sequence Homology Amino Acid biology Herbicides Genetic Complementation Test Arsenate Pteris Sequence Analysis DNA General Medicine biology.organism_classification Molecular biology Plant Leaves Arsenate reductase chemistry Biochemistry Mutation Pteris vittata Arsenates Agronomy and Crop Science Ars operon Triose-Phosphate Isomerase |
| Zdroj: | Plant Molecular Biology. 62:845-857 |
| ISSN: | 1573-5028 0167-4412 |
| DOI: | 10.1007/s11103-006-9060-8 |
| Popis: | Arsenic hyperaccumulator Pteris vittata L. (Chinese brake fern) grows well in arsenic-contaminated media, with an extraordinary ability to tolerate high levels of arsenic. An expression cloning strategy was employed to identify cDNAs for the genes involved in arsenic resistance in P. vittata. Excised plasmids from the cDNA library of P. vittata fronds were introduced into Escherichia coli XL-1 Blue and plated on medium containing 4 mM of arsenate, a common form of arsenic in the environment. The deduced amino acid sequence of an arsenate-resistant clone, PV4-8, had cDNA highly homologous to plant cytosolic triosephosphate isomerases (cTPI). Cell-free extracts of PV4-8 had 3-fold higher level of triosephosphate isomerase (TPI) specific activities than that found in E. coli XL-1 Blue and had a 42 kD fusion protein immunoreactive to polyclonal antibodies raised against recombinant Solanum chacoense cTPI. The PV4-8 cDNA complemented a TPI-deficient E. coli mutant. PV4-8 expression improved arsenate resistance in E. coli WC3110, a strain deficient in arsenate reductase but not in AW3110 deficient for the whole ars operon. This is consistent with the hypothesis that PV4-8 TPI increased arsenate resistance in E. coli by directly or indirectly functioning as an arsenate reductase. When E. coli tpi gene was expressed in the same vector, bacterial arsenate resistance was not altered, indicating that arsenate tolerance was specific to P. vittata TPI. Paradoxically, P. vittata TPI activity was not more resistant to inhibition by arsenate in vitro than its bacterial counterpart suggesting that arsenate resistance of conventional TPI reaction was not the basis for the cellular arsenate resistance. P. vittata TPI activity was inhibited by incubation with reduced glutathione while bacterial TPI was unaffected. Consistent with cTPI's role in arsenate reduction, bacterial cells expressing fern TPI had significantly greater per cent of cellular arsenic as arsenite compared to cells expressing E. coli TPI. Excised frond tissue infiltrated with arsenate reduced arsenate significantly more under light than dark. This research highlights a novel role for P. vittata cTPI in arsenate reduction. |
| Databáze: | OpenAIRE |
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