Submitted to: DNA Sequence
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/31/2005
Publication Date: 4/20/2005
Citation: Jordan, Robin H, Rickie B. Turley, Sherri L. Defauw, & Mark Steele. 2005. Characterization of a cDNA encoding metallothionein 3 from cotton (Gossypium hirsutum L.). DNA Sequence. 16(2):96-102. Interpretive Summary: The cotton crop is a valuable resource to the economy of the Mississippi Delta. We have identified a gene in cotton that produces a protein that binds copper. In plants, this protein appears to be beneficial in that it can act as a storage reservoir for copper (copper is required for plant growth). In other plants it can allow them to grow in toxic levels of copper. This protein appears to be important in cotton seedling development, especially after germination. One function may be to supply copper to the seedling as it emerges and becomes a photosynthetic (process in which plants produce food from sunlight) plant. This research is important to scientist because it allows them to study how proteins bind metals and how proteins increase the nutritional value of the seed by storing copper needed for essential processes in animal and humans
Technical Abstract: A cDNA encoding a metallothionein (MT) was isolated from a library constructed with polyA+ RNA purified from 48 h etiolated cotton (G. hirsutum L.) cotyledons. This cDNA encodes a deduced protein with 63 residues and a molecular weight of 6.3 kDa. The protein has 10 cysteines of which 4 are within the CXXCXCXXXXXC amino-terminus motif and six are within the CXCXXXCXCXXCXC carboxyl-terminus motif characteristic of the type III MT (MT3). Cotton MT3 protein sequence is 76.2, 69.8, 66.7 and 60.3% identical with MT3 from Carica papaya, Rubus idaeus, Ribes nigrum and Citrus unshiu, respectively. Cotton MT3 protein was also found to be 33.5% identical to Gossypium hirsutum type I MT. A fusion protein was constructed by producing PCR primers for the 5' and 3' ends of the cotton MT3 cDNA and ligating the PCR product inframe at the 3' end of a bacterial glutathione S-transferase (GST) gene in the pGEX3 vector. The 5' PCR primer incorporated a segment of the cotton MT3 noncoding region, resulting in an addition of 7 residues to the MT3 (after Factor Xa digestion) which increased the size of the expressed protein to 72 residues and 7.6 kDa. Expression of the 7.6 kDa protein in bacteria was confirmed by SDS-PAGE. Induction and accumulation of the GST-MT3 protein inhibited bacterial growth after 1 h. Addition of Cu (1 M to 1 mM), 1 mM cysteine, or 1 mM cystine to the media did not rescue growth. Additionally, this protein was evaluated for its ability to bind Cd, Cu, Ni and Zn in the bacterial expression system. We found that cotton MT3 preferably binds Cu, however, it may have lower selectivity to other metals tested.