Cloning and expression of the chloroplast copper/zinc-superoxide dismutase gene in upland cotton (Gossypium hirsutum L.).

Abstract

A full-length 1,043-base-pair cDNA clone encoding a chloroplast copper/zinc superoxide dismutase (Cu/Zn-SOD) of upland cotton was first isolated by rapid amplification of cDNA ends (RACE) from the leaves of the cotton (Gossypium hirsutum L.) variety 'CRI36'. Nucleotide sequence analysis of the clone revealed that it contained the complete coding sequence of the mature SOD isozyme subunit, along with a 60-amino acid transit peptide at N-terminal. The amino acid sequence predicted from the full-length clone showed 66%-74% homology with the amino acid sequences of Cu/Zn-SOD from several other plants. This gene was found to be expressed in the leaves and stems, but not in roots, flowers, and hypocotyls, indicating that the gene was expressed only in green tissues. Also, its expression was found to be most active at seedling stage and declined gradually in later development stages. Expression of this cotton Cu/Zn-SOD gene by using the pET-21a (+) expression vector in E. coli BL21 (DE3) led to the production of a novel 29 kD polypeptide with SOD enzyme activity, confirming that the cloned cotton Cu/Zn-SOD cDNA was indeed encoding a functioning SOD enzyme.