EXPRESSION OF TWO ISOFORMS OF THE ANTIFUNGALPROTEIN OSMOTIN FROM A COTTON GENE CLUSTER

摘要

Fungal diseases in cotton, such as various types of root rot and wilt, cause destruction of a large percentage of the cotton cropand large economic loss throughout the cottonbelt annually. Almost all plants naturally produce their own defensive proteins,called pathogenesis-related (PR) proteins, directed against pathogens such as fungi and bacteria. PR proteins called osmotins aremade in response to fungal pathogen stress or osmotic stress. The class of PR proteins called osmotins cause osmotic ruptureand destruction of pathogenic fungal cells by an unknown mechanism. Since osmotins have antifungal activity against mostfungal species, it may be possible to use the osmotin gene as a novel defense gene effective against numerous pathogenic fungi.To isolate prospective cotton osmotin genes to study osmotin gene structure, organization, and expression, genomic libraries inlambda phage were screened with a tobacco osmotin gene probe. Three overlapping genomic clones were found to encompass a29.0-kb cotton DNA segment encompassing a cluster of two genes and two pseudogenes. The two genes have an identity of92%, with open reading frames of 729 basepairs without introns, and encode putative preproteins of 242 amino acids. Two partialcDNA clones corresponding to the two genes were isolated from a cotton cDNA library, indicating that these genes are trulyexpressed in cotton. The two presumptive cotton osmotin preproteins can clearly be identified as members of the class of PR5proteins due to their identities with the deduced amino acid sequences of other PR5 preproteins. The two cotton osmotin preproteinswould have N-terminal signal sequences of 24 amino acids, and the mature forms of the proteins would likely be targetedfor extracellular secretion as a neutral isoforms. In addition to basal TATA and CAAT promoter elements, other prospectivepromoter elements, such as two ethylene response elements with the canonical sequence AGCCGCC, implicated as being positiveregulatory elements in the expression of a number of PR proteins, occur in the 5’-flanking sequences of the two genes. Thetwo pseudogenes are probably nonfunctional, since they have internal stop codons in their coding regions. When treated withethephon and hydrogen peroxide, cotton plants apparently are induced to express the osmotin proteins, as detected by Westernblot analysis with a polyclonal anti-osmotin antibody preparation.