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Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench]
Thesis (PhD)--University of Pretoria, 2010.
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University of Pretoria
2013
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| _version_ | 1867613679392915456 |
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| access_status_str | Open Access |
| author2 | Kunert, Karl J. |
| author_browse | Kunert, Karl J. |
| author_facet | Kunert, Karl J. |
| collection | Thesis |
| dc_rights_str_mv | © 2010 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Thesis (PhD)--University of Pretoria, 2010. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/28964 |
| institution | University of Pretoria (South Africa) |
| last_indexed | 2026-06-10T12:39:58.958Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/28964 Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] Kunert, Karl J. Chikwamba, Rachel K. andile.grootboom@dst.gov.za Grootboom, Andile W. Lysine-deficient kafirins Rnai down-regulation Sorghum UCTD Thesis (PhD)--University of Pretoria, 2010. Sorghum (Sorghum bicolor L. Moench) ranks fifth worldwide in production among cereals. It is a major staple food for millions in Africa and Asia, and a major livestock feed grain in developed countries. However, the sorghum grain is poor in lysine content, limiting its value as food and feed. In this study, I hypothesize that reduction of some of the major storage proteins that are inherently poor in lysine through in vitro manipulation will result in the enhanced expression of proteins with a better lysine profile and, thus, increased overall grain lysine content. Sorghum genotypes were screened for in vitro amenability and a sorghum genotype-tissue culture medium combination that yielded the highest somatic embryo callus formation and regeneration potential, was identified. This resulted in the establishment of a sorghum biolistic transformation method with a transformation efficiency of 3.36%, the highest reported to date. Using genetic engineering tools, the enhancement of the nutritional quality of grain sorghum was achieved by increasing the seed lysine content. An RNAi co-suppression strategy was employed and resulted in 45.23 and 77.55% increase in whole seed and endosperm lysine increase, respectively. The co-suppression RNAi constructs targeted the endosperm specific suppression of three lysine-poor storage proteins, namely ä-kaf-2, ã-kaf-1 and -2, and an enzyme that catalyzes seed lysine degradation, lysine keto-gluterate reductase (LKR). Seven independent transgenic events displayed successful transgene integration for both the selectable marker gene and the target constructs. However, the Southern blot hybridization analysis revealed two transgenic events that displayed transgene re-arrangement at the 5’promoter end, thus resulting in a lack of suppression of target proteins. Variations in target proteins co-suppression was observed with Western blot analysis and RT-PCR for both the target kafirins and LKR suppression, and no lysine improvement was observed where no kafirin suppression occurred. The transgenic co-suppression of the target kafirins resulted in the endosperm structural change from a hard, corneous endosperm to a soft, floury endosperm, consistent with ã-zein suppression in the Opaque-2 maize mutant. Plant Science unrestricted 2013-09-07T14:33:47Z 2010-10-24 2013-09-07T14:33:47Z 2010-09-02 2010-10-24 2010-10-23 Thesis Grootboom, AW 2010, Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench], PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://hdl.handle.net/2263/28964 > D10/713/ag http://hdl.handle.net/2263/28964 http://upetd.up.ac.za/thesis/available/etd-10232010-153005/ © 2010 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf application/pdf application/pdf application/pdf application/pdf application/pdf University of Pretoria |
| spellingShingle | Lysine-deficient kafirins Rnai down-regulation Sorghum UCTD Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] |
| title | Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] |
| title_full | Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] |
| title_fullStr | Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] |
| title_full_unstemmed | Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] |
| title_short | Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] |
| title_sort | effect of rnai down regulation of three lysine deficient kafirins on the seed lysine content of sorghum sorghum bicolor l moench |
| topic | Lysine-deficient kafirins Rnai down-regulation Sorghum UCTD |
| url | http://hdl.handle.net/2263/28964 http://upetd.up.ac.za/thesis/available/etd-10232010-153005/ |