[Cloning of a new catechol 1,2-dioxygenase gene (tfd C) from Plesiomonas and its expression in the E. coli].
A new catechol-1,2-dioxygenase gene (tfd C) was cloned from the Plesiomonas using the PCR method. Primers were designed according to the reported sequence of Catechol-1,2-dioxygenase (C120) gene from Alcaligenes eutroplus. The amplified fragment contained a 765 bp open reading frame (ORF), encoding a protein of 255 amino acids. The new tfd C gene shared a high homology with the one cloned from Alcaligenes eutroplus, showing only one base difference at 693 site (C-->A) and consequently one amino acid difference at 228 site (P-->T). The ORF was cloned to the plasmid pBluescriptII KS, which was transferred to E. coli JM109 and a positive clone, pBt2G, was then selected. A significant activity of C120 was detected in the positive clone. When the ORF was cloned to the plasmid pET-30a, which was transferred to E. coli BL21(DE3) plysS, the expected 33 kD protein was detected from a positive clone, pET30A, by SDS-PAGE. The C120 is a key enzyme in degrading aromatic pollutants in the environment. In order to use plants to degrade aromatic pollutants, the gene will be introduced into the turfgrass. To express the gene properly in plants, its translation initiation codon was modified from GTG to ATG. A similar activity of C120 was obtained following the modification.
Ma
Z
Z
Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433.
Luo
R
R
Xia
Y
Y
Wang
W
W
Chen
W
W
Kuai
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D050548:Catechol 1,2-Dioxygenase; D003001:Cloning, Molecular; D049308:Dioxygenases; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016981:Plesiomonas / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction
D010105:Oxygenases; D049308:Dioxygenases; D050548:Catechol 1,2-Dioxygenase