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KMID : 0578320090270040423
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Molecules and Cells
2009 Volume.27 No. 4 p.423 ~ p.428
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Molecular Cloning and Expression of a Cu/Zn-Containing Superoxide Dismutase from Thellungiella halophila
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Xu Xiaojing
Ren Dongtao
Zhou Yijun
Wei Shanjun
Yang Min
Bu Huahu
Kang Mingming
Wang Junli
Feng Jinchao
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Abstract
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Superoxide dismutases (SODs) constitute the first line of cellular defense against oxidative stress in plants. SODs generally occur in three different forms with Cu/Zn, Fe, or Mn as prosthetic metals. We cloned the full-length cDNA of the Thellungiella halophila Cu/Zn-SOD gene ThCSD using degenerate RT-PCR and rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the ThCSD gene (GenBank accession number EF405867) had an open reading frame of 456 bp. The deduced 152-amino acid polypeptide had a predicted molecular weight of 15.1 kDa, an estimated pI of 5.4, and a putative Cu/Zn-binding site. Recombinant ThCSD protein was expressed in Escherichia coli and assayed for SOD enzymatic activity in a native polyacrylamide gel. The SOD activity of ThCSD was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, confirming that ThCSD is a Cu/Zn-SOD. Northern blotting demonstrated that ThCSD is expressed in roots, stems, and leaves. ThCSD mRNA levels increased by about 30-fold when plants were treated with sodium chloride (NaCl), abscisic acid (ABA), and indole-acetic acid (IAA) and by about 50-fold when treated with UVB light. These results indicate that ThCSD is involved in physiological pathways activated by a variety of environmental conditions.
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KEYWORD
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expression, RACE, superoxide dismutase (SOD), ThCSD gene, Thellungiella halophila
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