Purification, characterization and cDNA cloning of the 200 kDa protein induced by cold acclimation in wheat.
We have purified to homogeneity the 200 kDa protein induced specifically by low temperature in wheat (Triticum aestivum L.). The boiling solubility of the protein has been used as a main step in the purification procedure. Amino acid composition indicates that the 200 kDa has a compositional bias for glycine (11.4%), threonine (13.3%), and alanine (22.0%). Using oligonucleotide probes, we have isolated a clone (p Wcs200) from a cold-acclimated winter wheat cDNA library. Northern analysis demonstrated that the expression of the corresponding gene was specifically upregulated by low temperature. Southern analysis showed that the gene organization and the relative copy number were identical in two cultivars differing in their capacity to develop freezing tolerance. Protein sequence and immunological analyses indicate that this protein shares similar features with the 50 kDa protein induced during cold acclimation of wheat. The two proteins are boiling-soluble, and possess similar repeated elements. These elements may be important for the development of freezing tolerance. We have shown that the 200 kDa protein is the largest member of a family of immunologically-related cold-induced proteins in wheat. Expression of pWcs200 in E. coli yielded a product of around 200 kDa, indicating that the clone contains most of the coding region for this protein.
Ouellet
F
F
Département des Sciences biologiques, Université du Québec à Montréal, Canada.
Houde
M
M
Sarhan
F
F
D000064:Acclimatization; D000595:Amino Acid Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003080:Cold Temperature; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D018076:DNA, Complementary; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D010940:Plant Proteins