Characterisation of two desiccation-linked dehydrins from Xerophyta humilis

 

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dc.contributor.advisor Farrant, Jill M en_ZA
dc.contributor.advisor Rafudeen, Suhail en_ZA
dc.contributor.author Fan, Cynthia en_ZA
dc.date.accessioned 2017-01-16T13:42:53Z
dc.date.available 2017-01-16T13:42:53Z
dc.date.issued 2016 en_ZA
dc.identifier.citation Fan, C. 2016. Characterisation of two desiccation-linked dehydrins from Xerophyta humilis. University of Cape Town. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/22723
dc.description.abstract In response to abiotic stresses, organisms throughout the plant kingdom, as well as microorganisms and micro-animals such as nematodes or tardigrades, have been observed to express Late Embryogenesis Abundant (LEA) proteins as protective mechanisms. However, despite two decades of research, little is understood about their physiological functions and this has led to extensive nomenclature, with a large amount of redundancy. The primary reason for this lack of insight into LEA protein functions is their highly hydrophilic and intrinsically disordered nature. Intrinsically disordered proteins (IDPs) cannot be studied using conventional methods of structural analyses such as X-ray crystallography and, therefore, alternative techniques are required. A combination of transgenic and in vitro studies have also shown that LEA proteins are most likely to behave as molecular chaperones by binding water and ions, preventing macromolecular aggregation and protecting enzymatic activity during dehydration. This study characterized two dehydrins that were expressed during dehydration in the desiccation tolerant plant, Xerophyta humilis. From a transcriptome analyses on X. humilis, cDNA for the two dehydrins were obtained. These sequences were first analysed using various in silico tools in order to identify putative dehydrin-specific characteristics. Subsequently, these two dehydrins were cloned and expressed for production of recombinant dehydrin protein. These proteins were then analysed in terms of structural and functional characteristics. Structurally, through the use of circular dichroism in an in vitro system, both dehydrins demonstrated the shift towards being increasingly alpha-helical when placed in environments of decreasing water content. The role of these two dehydrins in stabilizing enzymes during dehydration was subsequently investigated using citrate synthase (CS) and lactate dehydrogenase (LDH). The preservation of enzyme activity was observed in both CS and LDH. This preservation of enzyme activity was further maintained by the presence of trehalose. Anti-aggregation roles were also investigated, however, neither dehydrin demonstrated significant ability to minimize the aggregation of LDH. This study hopes to establish a pipeline for characterizing LEA proteins using structural and functional assays in order to provide alternative means of LEA protein classification. en_ZA
dc.language.iso eng en_ZA
dc.subject.other Molecular and Cell Biology en_ZA
dc.title Characterisation of two desiccation-linked dehydrins from Xerophyta humilis en_ZA
dc.type Master Thesis
uct.type.publication Research en_ZA
uct.type.resource Thesis en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Science en_ZA
dc.publisher.department Department of Molecular and Cell Biology en_ZA
dc.type.qualificationlevel Masters
dc.type.qualificationname MSc en_ZA
uct.type.filetype Text
uct.type.filetype Image
dc.identifier.apacitation Fan, C. (2016). <i>Characterisation of two desiccation-linked dehydrins from Xerophyta humilis</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/22723 en_ZA
dc.identifier.chicagocitation Fan, Cynthia. <i>"Characterisation of two desiccation-linked dehydrins from Xerophyta humilis."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2016. http://hdl.handle.net/11427/22723 en_ZA
dc.identifier.vancouvercitation Fan C. Characterisation of two desiccation-linked dehydrins from Xerophyta humilis. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2016 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/22723 en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Fan, Cynthia AB - In response to abiotic stresses, organisms throughout the plant kingdom, as well as microorganisms and micro-animals such as nematodes or tardigrades, have been observed to express Late Embryogenesis Abundant (LEA) proteins as protective mechanisms. However, despite two decades of research, little is understood about their physiological functions and this has led to extensive nomenclature, with a large amount of redundancy. The primary reason for this lack of insight into LEA protein functions is their highly hydrophilic and intrinsically disordered nature. Intrinsically disordered proteins (IDPs) cannot be studied using conventional methods of structural analyses such as X-ray crystallography and, therefore, alternative techniques are required. A combination of transgenic and in vitro studies have also shown that LEA proteins are most likely to behave as molecular chaperones by binding water and ions, preventing macromolecular aggregation and protecting enzymatic activity during dehydration. This study characterized two dehydrins that were expressed during dehydration in the desiccation tolerant plant, Xerophyta humilis. From a transcriptome analyses on X. humilis, cDNA for the two dehydrins were obtained. These sequences were first analysed using various in silico tools in order to identify putative dehydrin-specific characteristics. Subsequently, these two dehydrins were cloned and expressed for production of recombinant dehydrin protein. These proteins were then analysed in terms of structural and functional characteristics. Structurally, through the use of circular dichroism in an in vitro system, both dehydrins demonstrated the shift towards being increasingly alpha-helical when placed in environments of decreasing water content. The role of these two dehydrins in stabilizing enzymes during dehydration was subsequently investigated using citrate synthase (CS) and lactate dehydrogenase (LDH). The preservation of enzyme activity was observed in both CS and LDH. This preservation of enzyme activity was further maintained by the presence of trehalose. Anti-aggregation roles were also investigated, however, neither dehydrin demonstrated significant ability to minimize the aggregation of LDH. This study hopes to establish a pipeline for characterizing LEA proteins using structural and functional assays in order to provide alternative means of LEA protein classification. DA - 2016 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2016 T1 - Characterisation of two desiccation-linked dehydrins from Xerophyta humilis TI - Characterisation of two desiccation-linked dehydrins from Xerophyta humilis UR - http://hdl.handle.net/11427/22723 ER - en_ZA


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