Expression of synthetic maganin genes in potato

Abstract

This thesis describes the design and construction of four synthetic antimicrobial genes, their assembly into binary vectors and the subsequent Agrobacterium-mediated transformation of potato (Solanum tuberosum L.). Resulting plants were analysed at the molecular level for expression of the antimicrobial genes, and bioassays were performed to determine whether the synthetic genes conferred a disease resistance phenotype.

The synthetic genes selected for construction were based on the antimicrobial peptide magainin II. The peptide has activity against a range of bacteria, some fungi and protozoans, withouttoxic effects on higher eukaryotes. Magainin peptides disrupt microbial membranes by causing osmolysis, and are known to inhibit the in vitro growth of Erwinia carotovora, the causative agent of soft rot disease in potato.

The hypothesis that transgenic potato lines expressing synthetic magainin genes have improved resistance to microbial pathogens including Erwinia carotovora, the causative agent of soft rot diseases in potato, was investigated.

The coding sequence for magainin II, and three variants of magainin II, reported to either increase antimicrobial activity or increase resistance to plant protease cleavage were designed and constructed using potato codon preference. The magainin coding sequences were constructed from primers, and ligated into a chimeric gene for expression in plants. The four chimeric magainin genes were subsequently ligated into a binary vector for Agrobacterium-mediated transformation. Potato cultivar Iwa was transformed with these binary vectors to produce a population of independently derived transgenic plants for each vector.

The resulting transgenic plants were characterised the nucleic acid level. Southern analysis determined that the magainin genes had integrated into the potato genome. Reverse transcriptase-PCR (RT-PCR) analysis demonstrated that the majority of transgenic potato lines expressed the transgenes at the RNA level.

To determine whether the magainin peptides were being expressed at the protein level in transgenic potato lines, antibodies to the magainin peptide for use in western analysis were developed. The coding region of one of the magainin gene variants was ligated within the thioredoxin gene of the bacterial expression vector pET-32a. The resulting recombinant fusion gene was expressed in Escherichia coli, without cytotoxic effect. The thioredoxinmagainin fusion protein was purified and used to raise antibodies in rabbits. Reactive sera was produced by one rabbit, and was successfully used in western analysis to detect magainin peptides. A protocol was developed for extraction of transgene-derived cationic antimicrobial peptides from transgenic potato tissue. The extraction protocol was subsequently used with the sera in western analysis of protein from transgenic potato lines. Several potato lines were identified that expressed the magainin gene at the protein level.

A field trial with the transgenic potato plants was conducted, and harvested tubers were evaluated in bioassays for resistance to Erwinia carotovora ssp atroseptica. Results from bioassays identified a transgenic potato line with significantly improved resistance to soft rot compared with control lines. The same line was determined to have one of the highest levels of expression of the transgene-derived peptide. The result demonstrated that the design, construction and transformation of synthetic antimicrobial magainin genes was a successful strategy in introducing a novel form of disease resistance in potato plants.