Browsing by Author "Miller, Taryn Angela"

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    Insect bioactive capabilities of Epichloë festucae var lolii AR48 infected Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Miller, Taryn Angela
    As the modern world expands and develops, new innovative methodologies for more efficient and environmentally friendly agricultural practices are required. Loss of crops through abiotic (e.g. drought) and biotic (e.g. herbivory) stresses has a major effect on the success of an agricultural industry. For animal production pasture crops are a key aspect of animal husbandry and directly affects yield and health. Symbiotic fungi belonging to the genus Epichloë form associations with cool season forage grasses and have been exploited as a new innovative method for insect pest management. Ryegrass infected with the asexual E. festucae var lolii strain AR48 has insect bioactivity against both the stem boring fly (SBF-Ceradontha australis) and cutworm moth caterpillar (CC -Agrotis ipsilion). The bioactive/s targeting both insects is currently unknown. The aim of this thesis was to identify the gene/s and/or bioactive/s present in AR48 infected ryegrass that have bioactivity against the SBF and/or CC. Two approaches were taken; the known insect bioactive secondary metabolite pathways in Epichloë were investigated in AR48 through bioinformatics and mass spectrometry, and the gene ‘makes caterpillars floppy’ (mcf), encoding an insect toxin like protein, was investigated through reverse genetics and insect bioactivity trials. A new indole diterpene compound (IDT) was identified in AR48 infected plant material and this compound was absent in other Epichloë strains that do not have SBF and CC bioactivity. The same mcf gene allele as that present in the E. typhina mcf model, previously identified as having CC bioactivity, is present and predicted to be functional in AR48. The other Epichloë strains also have mcf genes predicted to be functional, however the mcf allele is different to the bioactive E. typhina mcf model. Overall, this project was able to identify a new IDT compound with potential insect bioactivity as well as identify two Epichloë mcf gene alleles that potentially have differing insect bioactivities.
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    Purification and characterisation of a secreted glycosidase, from the extreme xerophile Wallemia ichthyophaga : a thesis presented in partial fulfilment of the requirement for the degree in Master of Science in Biochemistry at Massey University Palmerston North, New Zealand
    (Massey University, 2014) Miller, Taryn Angela
    With recent pressure to reduce the environmental impact of leather production, research has been focused on the development of an alternative depilation method, as the conventional method for depilation contributes up to 60% of the total pollution produced. Contaminated salted ovine pelts stored at LASRA were easily depilated when drum washed, and the resultant leather was of good quality. The pelts were visibly contaminated with microorganisms, and it was thought that these may be secreting enzymes that loosened the wool fibre without damaging key structural skin components. Identification of the enzyme or enzymes was thus of interest. The microorganism/s responsible for the secretion of the depilation enzyme/s were isolated and identified through sequencing the 16S/18S ribosomal RNA genes. Depilation, using the crude secretome solutions, was then assessed using fresh ovine skin as well as SACPIC, a micro scale staining method used to assess skin structure. Unfortunately, none of the secretomes from either a single or a combination of the microorganisms isolated, had depilation activity. The secretome of W.ichthyophaga, a xerophilic filamentous fungus, which was consistently isolated from the contaminated pelts, was chosen to be characterised using proteomic methods. 1D SDS-PAGE gel/CHIP separation of the proteins in the secretome showed it contained mainly glycosidases, with no lipases, esterases, or proteases identified. Some of the proteins identified had suggested roles in resistance to osmotic pressure, while the remaining proteins were intracellular. Overall, 21 proteins were identified. A purification procedure involving AEX and SEC was successfully developed for the isolation of one of the glycosidases from the secretome. The resultant purified fractions formed a doublet band when analysed by SDS-PAGE. The reason for this remains unknown, but was shown not to be due to an impurity or heterodimerisation. The purified glycosidase was identifed as belonging to the GH3 family by mass spectrometry. It was found to have a pH optimum of pH 6.0, was optimally active at 10% NaCl, and was itself glycosylated. The glycosidase was able to hydrolyse both a- and ß- linked glycosidic bonds in di- and polysaccharides. Interestingly, both the disaccharide and artifical p-nitrophenol forms of galactose were not cleaved by the enzyme.