Mohan, Sara2012-07-092011https://hdl.handle.net/10182/4651Antimicrobial peptides (host defense peptides) are an evolutionarily conserved component of the innate immune response, and have been shown to kill or inhibit the growth of microbes such as bacteria, fungi, viruses, or parasites. Snakin-1 (SN1) and Snakin-2 (SN2) are low-molecular weight antimicrobial peptides produced in potato tubers. They share common structural features with more than 500 antimicrobial peptides discovered in plants and animals, such as an N-terminal putative signal sequence, a highly divergent intermediate region and a conserved cysteine-rich 60 amino-acid carboxyl-terminal domain. These proteins are thought to play important roles in the innate defense against invading microbes. SN1 and SN2 genes were isolated and sequenced from a range of potato cultivars/clones (Solanum tuberosum), as well as a diverse range of other Solanaceous species. The sequences were aligned with reference sequences of the StSN1 gene (AJ320185) and the StSN2 gene (AJ312424) using Vector NTI Advance 10 software. Allelic variation both within and between genotypes and/or species was observed for both SN1 (four alleles) and SN2 (six alleles). Most of the variation occurred within introns, with relatively few nucleotide substitutions in exons. The predicted amino acid sequence was highly conserved; identical for all SN1 alleles, with only minor amino acid substitutions observed in SN2 alleles present in different Solanum species. Overexpression of the SN genes in potato was achieved using Agrobacterium-mediated gene transfer. Plant expression vectors with both sense and antisense orientations of SN1 (a1 allele) and SN2 (b1 allele) genes were constructed under the regulatory controls of the potato light inducible Lhca3 gene. Potato plants, cultivar Iwa, were transformed via Agrobacterium tumefaciens harboring binary vector containing the intragenic SN cassettes. The resulting plants were confirmed as being transgenic by PCR, then analysed at the molecular level for expression of the transgenes. Quantitative Reverse Transcriptase-PCR (qRT-PCR) analysis demonstrated that the majority of the transgenic potato lines overexpressed the SN genes at RNA level. Based on qRT-PCR of each SN gene, seven and eight lines respectively were selected from each of the Lhca3-SN1 and Lhca3-SN2 transgenic plant populations for further characterisation. Overexpression of the SN genes in leaves, stems, roots and tubers was determined by qRT-PCR analysis and these selected transgenic lines were evaluated in bioassays for resistance to Pectobacterium atrosepticum (formally Erwinia carotovora subsp. atroseptica) causal agent of blackleg in potatoes. Results from the pathogenicity bioassays showed that overexpression of either the SN1 gene or the SN2 gene from the intragenic cassettes conferred resistance to P. atrosepticum blackleg disease. This result has established the functional role of SN genes in plant defense against pathogens in potato. Antisense transformants were difficult to recover and failed to regenerate plants. This indicates that these highly conserved Snakin proteins may also play an important role in cellular metabolism other than just defense against invading microorganism.x, 182 pagesenSnakin-2allelespotatoSnakin-1Agrobacterium-mediated transformationPectobacterium atrosepticumErwinia carotovora subsp. atrosepticaqRT-PCRblacklegLhca3 promoterThesisQ112887280