The characterization of vacuolar pyrophosphatase expression in sugarcane

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dc.contributor.advisorGroenewald, J-H.
dc.contributor.advisorBotha, F. C.
dc.contributor.authorSwart, Johannes Cornelius
dc.contributor.otherUniversity of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)
dc.date.accessioned2008-07-31T12:19:28Zen_ZA
dc.date.accessioned2010-06-01T08:32:31Z
dc.date.available2008-07-31T12:19:28Zen_ZA
dc.date.available2010-06-01T08:32:31Z
dc.date.issued2005-03
dc.descriptionThesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005.
dc.description.abstractVacuolar Pyrophosphatase (V-PPase) has never been studied in sugarcane before and to date nothing is known about V-PPase in sugarcane, except for the sequences of a few expressed sequence tags (ESTs). The aim of this project was to characterize V-PPase expression in several hybrid sugarcane varieties that differ significantly in sucrose content, with the main objective of the study to assess whether V-PPase is correlated in any way to the sucrose storage phenotype. Therefore, the goals of this project were to (i) develop molecular tools for the detection and quantification of V-PPase on a DNA, RNA, protein and enzyme level and (ii) to use these tools to characterize the expression of V-PPase within the culm of the three hybrid varieties. The cDNA sequence of the catalytic subunit of the sugarcane V-PPase gene was cloned, expressed in a bacterial system and the V-PPase peptide was purified. This peptide was used for the immunization of mice and the production of polyclonal anti-VPPase antiserum. Anti-VPPase antiserum reacted specifically with a single polypeptide among vacuolar membrane proteins. Moreover, anti-VPPase antiserum recognized V-PPase from various monocotyledons and dicotyledons. The anti-VPPase antiserum was used for the establishment of an ELISA system to determine V-PPase protein content in vacuolar membrane preparations. This system proved to have several advantages over the protein blotting technique and shared a strong linear relation with V-PPase specific activity, showing that these two tests are compatible and reliable. The optimisation of sugarcane V-PPase zero-order kinetics was fundamental in order to measure V-PPase specific activity accurately. It had a relative broad pH optimum, retaining more than 90% of its maximum activity between pH 6.50 and 7.25. V-PPase required both Mg2+ and K+, in addition to PPi, for maximum activity in vitro. The reported kinetic variables are within range of previous data determined for other species, including mung bean, red beet and sugar beet. V-PPase protein level and specific activity within the sugarcane culm followed a similar trend , withoiofofoenaobserved for sucrose accumulation rates observed in sugarcane. Moreover, V-PPase protein contents and specific activity share the same general trend as total sucrose content in a specific tissue compared among the three varieties. No significant differences were observed in V-ATPase activity among the three varieties. Our findings suggest that V-PPase may play a role in sucrose accumulation in sugarcane.en
dc.identifier.urihttp://hdl.handle.net/10019.1/1756
dc.language.isoen
dc.publisherStellenbosch : University of Stellenbosch
dc.rights.holderUniversity of Stellenbosch
dc.subjectDissertations -- Plant biotechnologyen
dc.subjectTheses -- Plant biotechnologyen
dc.subjectPyrophosphatesen
dc.subjectPlant vacuolesen
dc.subjectTonoplastsen
dc.subjectSugarcane -- Geneticsen
dc.subjectSucrose -- Metabolismen
dc.titleThe characterization of vacuolar pyrophosphatase expression in sugarcaneen
dc.typeThesis
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