Purification, Characterization and Molecular Cloning of a Corresponding PCR Product and cDNA Transcript of a Wound Inducible Tomato Leaf Protein

Abstract

Tomato plants respond to wounding caused by herbivorous insects with the induction of certain defensive genes. This wound response is mediated by systemin, an 18 amino acid polypeptide derived from a larger precursor called prosystemin. Transgenic plants that overexpress the systemin precursor exhibit a constitutive wound response allowing the accumulation of high levels of proteins produced by these defensive genes. Reported here is the purification of one of these defense related proteins from transgenic plant extracts by polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was obtained after blotting to PVDF-membranes. An oligonucleotide corresponding to the amino acid sequence was then designed and used as a primer in the polymerase chain reaction (PCR). Three specific PCR products were obtained. The largest one (900 bp) was used to screen a tomato leaf cDNA library. A cDNA clone of 2.1 kb was retrieved. Preliminary sequence data has identified the clone as a ketol-acid reductoisomerase. The second largest PCR product (500 bp) was cloned via a plasmid vector and preliminary sequence data has identified it as an acyl-CoA binding protein. The role that these proteins play in the systemic wound-response remains to be found.