ABSTRACT
Cassava is currently the second most important source of carbohydrates on the African
continent. In the last two decades, cassava crops have been severely affected by
outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus
(SACMV) has been associated with CMD outbreaks in the Mpumalanga province.
Advances in post-transcriptional gene silencing (PTGS) technology have provided
promising new strategies for the engineering of virus resistance in plants. Inverted repeat
(IR) constructs are currently the most potent inducers of PTGS, however, these constructs
are inherently unstable. The purpose of this study was to develop IR constructs with an
improved stability for the efficient induction of PTGS in plants. Two mismatched
inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other
targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully
created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the
constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize
the linear conformation of the IR constructs, as they were efficiently propagated by E.coli
DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found
that the number of mismatches on the BC1 construct (17.5%) was ideal, as the
subsequent stability of the predicted RNA hairpin was not affected. Due to the higher
number of mismatches on the AC1 construct (23.5%), it was found that the loop region of
the RNA hairpin was marginally destabilized. Despite this, long stretches of stable
dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS.
Interestingly, it was observed that the mismatched IR constructs, although still replicated
in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that
the stability of a mismatched IR construct may be influenced by the particular
intracellular environment of an organism. Due to the recalcitrance of cassava to
transformation, a model plant system, Nicotiana benthamiana, was used to screen
constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated
transformation and regeneration of N. benthamiana was optimized, and 86%
transformation efficiency was achieved when using leaf disk explants. It was found that
the addition of an ethylene scrubber, potassium permanganate, substantially increased the
rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene
iv
integration was confirmed by PCR amplification of the hptII gene in the T-DNA region.
Transgene expression was confirmed by screening for GUS and GFP reporter genes. No
toxic responses to the transgene have been observed thus far. Studies are currently
underway to confirm the stability of the mismatched IR constructs in N. benthamiana.
PAGE Northern blotting is being done, as the detection of siRNAs derived from the
transgene will confirm that constructs are functional. In addition, infectivity assays are
underway to determine the efficacy of BC1 knockdown by a stably integrated construct.
Due to the enhanced stability of mismatched IR constructs, they may be an appealing
alternative to currently available intron-spliced, or exact matched hairpin systems.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/4685 |
| Date | 19 March 2008 |
| Creators | Harmse, Johan |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 11486195 bytes, application/pdf, application/pdf |
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