Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Robert W. Thornburg
A potato Proteinase Inhibitor II gene (pin2T) was isolated and characterized both at the molecular level and the functional level. The open reading frame as well as the 5[superscript]' and 3[superscript]' flanking regions were sequenced. The coding region and 3[superscript]' flanking region showed 86% and 87% identity with those of the previously isolated wound-inducible potato Proteinase Inhibitor IIK gene (pin2K) sequence, respectively. However, the 5[superscript]' flanking region of the pin2T is highly homologous (91% identity) with that of pin2K from -767 to +29, relative to the transcription start site of the wound-inducible pin2K. The 5[superscript]' flanking region of pin2T was linked to the reporter gene (chloramphenicol acetyl transferase, CAT; or [beta]-glucuronidase, GUS) coding sequences in constructions that contained the terminator from the wound-inducible pin2K gene. The chimeric genes were transferred to tobacco plants using Agrobacterium tumefaciens. The presence of the constructions in the transgenic tobacco plants was confirmed by polymerase chain reaction (PCR). Expression of CAT or GUS activities in transgenic plants driven by pin2T promoter indicated that pin2T is not a wound-inducible gene. GUS activities driven by promoter deletion mutants ruled out the presence of the silencer sequences in upstream portion of the pin2T promoter that differed from pin2K. Comparison of pin2T promoter sequence with that of the wound-inducible pin2K indicates that there are four small deletions which are located at -221 to -200, -263 to -254, -523 to -462, and -759 to -708 relative to the transcription start site of pin2K. When these deleted sequences are searched through Genebank, we identified one sequence that is found three times in pin2K and is completely deleted from pin2T. This sequence, 5[superscript]'-AGTAAA-3[superscript]', is found in a wide variety of other wound-inducible genes but is not easily found in the published promoter sequences of the other plant genes. Nuclear proteins from unwounded and wounded potato leaves bound to the proximal promoter region, downstream of the 5[superscript]'-AGTAAA-3[superscript]', of pin2T. A third deletion located at -523 to -462 relative to the transcription initiation site of pin2K, was moderately homologous (72% identity) with a putative sucrose-responsive element of sucrose-inducible genes. Deletion of this sequence was correlated with a loss of sucrose inducibility in the pin2T promoter. CAT activity driven by pin2T was not enhanced by sucrose, while sucrose enhances the expression of pin2K-CAT construct in transgenic tobacco plants. In addition to the sucrose effect on the chimeric gene expression, the effects of plant hormones, abscisic acid (ABA) and [alpha]-naphthalene acetic acid ([alpha]-NAA), were examined in transgenic tobacco leaves and calli, respectively. CAT activity driven by the non-wound-inducible pin2T promoter was not induced by ABA or derepressed in the absence of [alpha]-NAA as was the wound-inducible pin2K promoter.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Park, Sanggyu, "Characterization of a potato Proteinase Inhibitor II gene whose expression in not wound-responsive " (1991). Retrospective Theses and Dissertations. 9566.