Characterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosa

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dc.contributor.authorConrad, Nailahen_ZA
dc.date.accessioned2014-07-30T17:36:16Z
dc.date.available2014-07-30T17:36:16Z
dc.date.issued2005en_ZA
dc.descriptionIncludes bibliographical references.
dc.description.abstractXerophyta viscosa (Baker) is a resurrection plant with the ability to survive desiccation and rehydrate upon watering with minimal tissue damage. XVEF was isolated by differential screening of aX viscosa dehydration stress cDNA library. Reconstruction of the full length XVEF cDNA was conducted utilizing overlap extension PCR. XVEF codes for a putative calcium-binding protein and sequence analysis indicated that it has a 708 bp ORF corresponding to a protein with a molecular mass of26.95 kDa, has a conserved calcium-binding EF-hand motif, potential phosphorylation sites, a pI of 6.49 and a putative conserved transmembrane domain spanning residues 90-107. Northern blot analyses of total RNA indicated that XVEF transcript increased 48 hours after 100/-lM ABA application and was present between 12 and 48 hours in response to a low temperature stress (4°C). A second gene, XvCaM, was isolated from a low temperature (4°C) cDNA stress library. Sequence analysis indicates that it has a 450 bp ORF corresponding to a 16.39 kDa protein, a pI of 3.90 and potential phosphorylation sites. It apparently encodes a calcium-binding protein with putatively three EF-hands which showed the highest similarity to plant calmodulins. XVCAM was heterologously expressed in E. coli as a fusion protein with a 6X His-tag and it was shown to be a functional protein that binds Ca2+ by utilizing a 45CaCh overlay assay. Northern blot analyses of XvCaM using total RNA under low (4°C) and high temperature treatment (42°C) showed constitutive expression levels of the transcript. Under the dehydration/rehydration treatment transcript levels decreased between 40% R WC dehydration and 26% RWC rehydration. The northern blot conducted with polysomal RNA isolated from 150 mM NaCI treatment also showed constitutive expression. Western blot analyses using anti-XVCAM polyclonal antibodies showed that the protein accumulated at 24 hours during the NaCI stress and at 15% RWC (dry) to 40% RWC (rehydration) during the dehydration/rehydration stress. The northern and western analyses results suggest that XVCAM undergoes post-translational modifications and XvCaM mRNA is possibly stored for rapid recovery processes upon rehydration. These results indicate that XVEF and XvCaM are possibly calcium-binding proteins most likely involved in modulating stress-responsive calcium-signaling cascades.en_ZA
dc.identifier.apacitationConrad, N. (2005). <i>Characterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosa</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/4251en_ZA
dc.identifier.chicagocitationConrad, Nailah. <i>"Characterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosa."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2005. http://hdl.handle.net/11427/4251en_ZA
dc.identifier.citationConrad, N. 2005. Characterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosa. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Conrad, Nailah AB - Xerophyta viscosa (Baker) is a resurrection plant with the ability to survive desiccation and rehydrate upon watering with minimal tissue damage. XVEF was isolated by differential screening of aX viscosa dehydration stress cDNA library. Reconstruction of the full length XVEF cDNA was conducted utilizing overlap extension PCR. XVEF codes for a putative calcium-binding protein and sequence analysis indicated that it has a 708 bp ORF corresponding to a protein with a molecular mass of26.95 kDa, has a conserved calcium-binding EF-hand motif, potential phosphorylation sites, a pI of 6.49 and a putative conserved transmembrane domain spanning residues 90-107. Northern blot analyses of total RNA indicated that XVEF transcript increased 48 hours after 100/-lM ABA application and was present between 12 and 48 hours in response to a low temperature stress (4°C). A second gene, XvCaM, was isolated from a low temperature (4°C) cDNA stress library. Sequence analysis indicates that it has a 450 bp ORF corresponding to a 16.39 kDa protein, a pI of 3.90 and potential phosphorylation sites. It apparently encodes a calcium-binding protein with putatively three EF-hands which showed the highest similarity to plant calmodulins. XVCAM was heterologously expressed in E. coli as a fusion protein with a 6X His-tag and it was shown to be a functional protein that binds Ca2+ by utilizing a 45CaCh overlay assay. Northern blot analyses of XvCaM using total RNA under low (4°C) and high temperature treatment (42°C) showed constitutive expression levels of the transcript. Under the dehydration/rehydration treatment transcript levels decreased between 40% R WC dehydration and 26% RWC rehydration. The northern blot conducted with polysomal RNA isolated from 150 mM NaCI treatment also showed constitutive expression. Western blot analyses using anti-XVCAM polyclonal antibodies showed that the protein accumulated at 24 hours during the NaCI stress and at 15% RWC (dry) to 40% RWC (rehydration) during the dehydration/rehydration stress. The northern and western analyses results suggest that XVCAM undergoes post-translational modifications and XvCaM mRNA is possibly stored for rapid recovery processes upon rehydration. These results indicate that XVEF and XvCaM are possibly calcium-binding proteins most likely involved in modulating stress-responsive calcium-signaling cascades. DA - 2005 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2005 T1 - Characterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosa TI - Characterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosa UR - http://hdl.handle.net/11427/4251 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/4251
dc.identifier.vancouvercitationConrad N. Characterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosa. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2005 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/4251en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherCell Biologyen_ZA
dc.titleCharacterization of XVEF and XvCaM : two calcium-binding proteins isolated from the resurrection plant Xerophyta viscosaen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMScen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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