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Masters Degrees (Botany)

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Browsing Masters Degrees (Botany) by Author "Cassim, Tasmien Nadine."

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    Comparision of two promoters driving transgene expression in water-stressed sugarcane.
    (1999) Cassim, Tasmien Nadine.; Huckett, Barbara Isobel.; Botha, Frikkie Coenraad.; Watt, Maria Paula Mousaco Deoliveira.
    For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites.