Molecular cloning and analysis of a cotton gene cluster of two genes and two pseudogenes for the PR5 protein osmotin

To isolate prospective cotton osmotin genes to study their gene structure, organization, and expression, cotton genomic libraries in lambda phage were screened using tobacco and cotton osmotin gene probes. Three overlapping clones encompassing a 29.0-kb cotton DNA segment were found to contain a cluster of two genes and two pseudogenes. The two genes have an identity of 92%, with open reading frames of 729 basepairs without introns, and would encode conceptual preproteins of 242 amino acids. Two partial cDNA clones corresponding to the two genes were isolated from a cotton cDNA library, indicating that the genes are indeed expressed in cotton. The two presumptive cotton osmotin preproteins can clearly be classified as PR5 proteins due to their identities with the deduced amino acid sequences and predicted three-dimensional structures of other PR5 preproteins. The two osmotin preproteins have predicted N-terminal signal sequences of 24 amino acids, and the mature forms of the proteins might be targeted for extracellular secretion as neutral isoforms. Prospective promoter elements, such as two ethylene response elements, implicated as being positive regulatory elements for expression of other PR proteins, occur in the 5′-flanking sequences of the two genes. The two pseudogenes are likely nonfunctional, because they have internal stop codons in their coding regions. Cotton plants are apparently induced to express the osmotin proteins upon treatment with ethephon and hydrogen peroxide, as detected by Western blot analysis with a polyclonal anti-osmotin antibody preparation.