Molecular cloning and expression of a Cu/Zn-Containing superoxide dismutase from Thellungiella halophila.
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.
Xu
Xiaojing
X
Department of Biological Sciences, College of Life and Environmental Sciences, Central University for Nationalities, Beijing, China. xuxiaojing@cun.edu.cn
Zhou
Yijun
Y
Wei
Shanjun
S
Ren
Dongtao
D
Yang
Min
M
Bu
Huahu
H
Kang
Mingming
M
Wang
Junli
J
Feng
Jinchao
J
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D019607:Brassicaceae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics*
D010940:Plant Proteins; D011994:Recombinant Proteins; D013482:Superoxide Dismutase