Molecular cloning and expression of a Cu/Zn-Containing superoxide dismutase from Thellungiella halophila

• Published in: Molecules and cells 2009, 27(4), , pp.423-428
• Main Authors: Xu, Xiaojing (Central University for Nationalities, Beijing, China), E-mail: [e-mail], Zhou, Yijun (Central University for Nationalities, Beijing, China), Wei, Shanjun (Central University for Nationalities, Beijing, China), Ren, Dongtao (China Agricultural University, Beijing, China), Yang, Min (Central University for Nationalities, Beijing, China), Bu, Huahu (Central University for Nationalities, Beijing, China), Kang, Mingming (Central University for Nationalities, Beijing, China), Wang, Junli (Central University for Nationalities, Beijing, China), Feng, Jinchao (Central University for Nationalities, Beijing, China)
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biochemistry; Biomedical and Life Sciences; Biomedicine; Biotechnology; Blotting, Northern; Brassicaceae - enzymology; Brassicaceae - genetics; Cell Biology; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; expression; Gene Expression; Genes, Plant; Humans; Life Sciences; Molecular Sequence Data; Plant Proteins - biosynthesis; Plant Proteins - genetics; Plant Roots - enzymology; Plant Roots - genetics; RACE; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; SUPEROXIDE DISMUTASE; Superoxide Dismutase - biosynthesis; Superoxide Dismutase - genetics; SUPEROXIDO DISMUTASA; SUPEROXYDE DISMUTASE; ThCSD gene; Thellungiella halophila; 생물학
• ISSN: 1016-8478; 0219-1032
• DOI: 10.1007/s10059-009-0060-9

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.