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The characterization of vacuolar pyrophosphatase expression in sugarcane
Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : University of Stellenbosch
2008
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| _version_ | 1867613926090342400 |
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| access_status_str | Open Access |
| author | Swart, Johannes Cornelius |
| author2 | Groenewald, J-H. |
| author_browse | Groenewald, J-H. Swart, Johannes Cornelius |
| author_facet | Groenewald, J-H. Swart, Johannes Cornelius |
| author_sort | Swart, Johannes Cornelius |
| collection | Thesis |
| dc_rights_str_mv | University of Stellenbosch |
| description | Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/1756 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:43:54.041Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2008 |
| publishDateRange | 2008 |
| publishDateSort | 2008 |
| publisher | Stellenbosch : University of Stellenbosch |
| publisherStr | Stellenbosch : University of Stellenbosch |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/1756 The characterization of vacuolar pyrophosphatase expression in sugarcane Swart, Johannes Cornelius Groenewald, J-H. Botha, F. C. University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB) Dissertations -- Plant biotechnology Theses -- Plant biotechnology Pyrophosphates Plant vacuoles Tonoplasts Sugarcane -- Genetics Sucrose -- Metabolism Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. Vacuolar 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. Masters 2008-07-31T12:19:28Z 2010-06-01T08:32:31Z 2008-07-31T12:19:28Z 2010-06-01T08:32:31Z 2005-03 Thesis http://hdl.handle.net/10019.1/1756 en University of Stellenbosch application/pdf Stellenbosch : University of Stellenbosch |
| spellingShingle | Dissertations -- Plant biotechnology Theses -- Plant biotechnology Pyrophosphates Plant vacuoles Tonoplasts Sugarcane -- Genetics Sucrose -- Metabolism Swart, Johannes Cornelius The characterization of vacuolar pyrophosphatase expression in sugarcane |
| title | The characterization of vacuolar pyrophosphatase expression in sugarcane |
| title_full | The characterization of vacuolar pyrophosphatase expression in sugarcane |
| title_fullStr | The characterization of vacuolar pyrophosphatase expression in sugarcane |
| title_full_unstemmed | The characterization of vacuolar pyrophosphatase expression in sugarcane |
| title_short | The characterization of vacuolar pyrophosphatase expression in sugarcane |
| title_sort | characterization of vacuolar pyrophosphatase expression in sugarcane |
| topic | Dissertations -- Plant biotechnology Theses -- Plant biotechnology Pyrophosphates Plant vacuoles Tonoplasts Sugarcane -- Genetics Sucrose -- Metabolism |
| url | http://hdl.handle.net/10019.1/1756 |
| work_keys_str_mv | AT swartjohannescornelius thecharacterizationofvacuolarpyrophosphataseexpressioninsugarcane AT swartjohannescornelius characterizationofvacuolarpyrophosphataseexpressioninsugarcane |