Chemical synthesis, cloning and expression of a gene encoding systemin, a proteinase inhibitor-inducing factor
Master Thesis
1996
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University of Cape Town
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Abstract
Wound-inducible proteinase inhibitors in plants elicit a defence mechanism by inactivating the proteinases of insects. This triggers a feedback mechanism causing overproduction of digestive enzymes together with a decrease in appetite, leading to starvation. System in, a polypeptide proteinase inhibitor-inducing factor, when applied to cut stems of young tomato plants induces the accumulation of inhibitors in a manner similar to the normal wounding response. We designed and synthesised the minus strand oligonucleotide template complementary to the system in DNA sequence using Escherichia coli codon preferences. The double stranded fragment encoding the 18 amino acid residue systemin was cloned into pUCJ 8 for amplification and subcloning into pMAL-pk for expression as a maltose binding-fusion protein. The recombinant systemin was released by enterokinase and isolated by HPLC. After further purification, the physical characteristics including amino acid composition, peptide sequence and molecular weight of r-systemin were determined. When the recombinant peptide was applied to young tomato plants, it induced the accumulation of proteinase inhibitor I messenger RNA.
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Bibliography: leaves 116-120.
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Ma, P. 1996. Chemical synthesis, cloning and expression of a gene encoding systemin, a proteinase inhibitor-inducing factor. University of Cape Town.