Document Type Doctoral Thesis Author Grootboom, Andile W URN etd-10232010-153005 Document Title Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench] Degree PhD Department Plant Science Supervisor
Advisor Name Title Prof K Kunert Co-Supervisor Dr R Chikwamba Supervisor Keywords
- lysine-deficient kafirins
- RNAi down-regulation
Date 2010-09-02 Availability unrestricted AbstractSorghum (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 š-kaf-2, „-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 5ípromoter 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 „-zein suppression in the Opaque-2 maize mutant.
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Please cite as follows:
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://upetd.up.ac.za/thesis/available/etd-10232010-153005/ >
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28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access 00front.pdf 149.74 Kb 00:00:41 00:00:21 00:00:18 00:00:09 < 00:00:01 01chapters1-2.pdf 518.62 Kb 00:02:24 00:01:14 00:01:04 00:00:32 00:00:02 02chapter3.pdf 337.29 Kb 00:01:33 00:00:48 00:00:42 00:00:21 00:00:01 03chapters4-5.pdf 688.68 Kb 00:03:11 00:01:38 00:01:26 00:00:43 00:00:03 04annexure.pdf 82.03 Kb 00:00:22 00:00:11 00:00:10 00:00:05 < 00:00:01