Characterization and Expression of a Wound-Inducible Proteinase Inhibitor II Gene from Potato
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An 8-kb fragment of potato DNA containing the potato Inhibitor II gene (called Inhibitor IIK gene) was isolated from a library of EcoRI-restricted potato DNA in λ bacteriophage. A 2.6-kb TaqI fragment containing the gene was subcloned into the plasmid pUC13 and sequenced. This gene is one of 10 to 12 Inhibitor II genes identified by analysis of fragments from restriction enzyme digests of total Russet Burbank genomic DNA by hybridization with nick-translated tomato Inhibitor II complementary DNA (cDNA). A 1.0-kb fragment from the 3’ region of the gene, containing only 11 bp of the open reading frame, hybridized strongly with mRNA species from wounded leaves, but only weakly with mRNA from unwounded leaves or tubers. This indicated that the gene is a wound-inducible gene. Approximately 900 bases from the 5’ region of the gene and 1,000 bases from the 3’ region were fused with the open reading frame of the reporter gene encoding the enzyme chloramphenicol acetyl transferase (CAT) gene. The chimeric gene was cloned into a binary transformation vector derived from the Ti plasmid and successfully used to transform tobacco plants with a wound-inducible CAT gene. Thus, nucleotide sequences in the 5’ and 3’ regions of the Inhibitor IIK gene are sufficient for wound-inducible expression of CAT activity. These experiments also demonstrate that biochemical components present in cells of wounded tobacco can recognize the potato Inhibitor IIK regulation sequences and can activate the expression of the CAT gene.
KeywordsChloramphenicol Acetyl Transferase Wounded Leaf Transform Tobacco Plant Chloramphenicol Acetyl Transferase Activity Potato Inhibitor
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- 2.An, G., B.D. Watson, and B.H. Howard (1982) Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells. Mol. Cell. Biol. 2: 1044–1051.Google Scholar
- 6.Feeny, P. (1976) Plant apparency and chemical defense. In Recent Advances in Phytochemistry, Vol. 10, J.W. Wallace and R.L. Mansell, eds. Plenum Press, New York, pp. 1–40.Google Scholar
- 9.Graham, J.S., G. Hall, G. Pearce, and C.A. Ryan (1986) Regulation of synthesis of proteinase inhibitors I and II mRNAs in leaves of wounded tomato plants. Planta (in press).Google Scholar
- 13.Lee, J.S., W.E. Brown, J.S. Graham, G. Pearce, E. Fox, T. Dreher, K. Ahern, G.D. Pearson, and C.A. Ryan (1986) Molecular characterization and phylogenetic studies with a wound-inducible proteinase inhibitor gene in Lycopersicon species. Proc. Natl. Acad. Sci., USA (in press).Google Scholar
- 14.Maniatis, T., I. Fritsch, and J. Sambrook, eds. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
- 18.Riven, C.J., E.A. Zimmer, and V. Walbot (1982) Isolation of DNA and DNA recombinants from maize. In Maize for Biological Research, W.F. Sheridan, ed. Plant Molecular Biology Association, Charlottesville, Virginia, pp. 161–164.Google Scholar
- 20.Ryan, C.A. (1981) Proteinase inhibitors. In The Biochemistry of Plants, Vol. 6, P.K. Stumpf and E.E. Conn, eds. Academic Press, Inc., New York, pp. 351–370.Google Scholar
- 22.Ryan, C.A., G. Pearce, G. An, and R.W. Thornburg (1986) The regulation of expression of proteinase inhibitor genes in food crops. Acta Horticulturae (in press).Google Scholar
- 23.Thornburg, R.W., G. An, T.E. Cleveland, R. Johnson, and C.A. Ryan (1986) Wound-inducible expression of potato Inhibitor II gene in transgenic tobacco plants. Proc. Natl. Acad. Sci., USA (in press).Google Scholar