Isolation and characterisation of a culm-specific promoter element from sugarcane

Goshu Abraha, Tsion (2005-03)

Thesis (MSc (Botany and Zoology. Plant Biotechnology))--University of Stellenbosch, 2005.


Sugarcane (Saccharum spp) is an important crop worldwide and is cultivated for the high level of sucrose in its mature internodes. Because of the exhaustion of the genetic potential in the commercial sugarcane germplasm conventional breeding has not lately been able to enhance sucrose content. Currently there is a concerted effort to improve culm sucrose content by genetic engineering which will require appropriate transgenes and promoters. One of the major constraints to genetic engineering of sugarcane is the lack of stable promoters required to drive tissue- or organ-specific expression of transgenes. Tissue and developmental stage specific promoters allow targeting of transgene activity and in doing so reduce the impact on non-target tissues. These promoters could also be advantageous to manipulate certain aspects of sucrose metabolism specifically in mature culm tissue. In addition, no promoters are currently freely available to the South African Sugar Industry for use in their transgenic program. The primary goal of this project was therefore to isolate a mature tissue-specific promoter for use in transgenic sugarcane plants. The approach followed was firstly, to identify an endogenous gene expressed in the desired pattern, and then to isolate the corresponding promoter from the sugarcane genome. cDNA macroarrays were initially used to identify differentially expressed sequences. The tissue specificity of potential clones was confirmed using RNA blot analysis. Two clones (c23-a and c22-a) were isolated and confirmed to be mature culm specific. Clone c22-a (putative dirigent-like protein) was selected for promoter isolation based on its culm tissue specific expression pattern and its proximity to the 5’ end of the gene. Furthermore, to confirm the activity of this promoter in the storage parenchyma cells, the exact cellular localisation of the transcript in the mature tissue was determined through in situ hybridisation. In situ hybridisation results confirmed the presence of the transcript in the parenchyma cells of mature culm tissue only. Moreover, the transcript is present in high concentrations in the parenchyma tissues surrounding the vascular bundles and parenchyma cells of the vascular complex. The selected dirigent-like gene was sequenced to allow the design of primers that could be used for the isolation of the corresponding promoter region using a long-range inverse PCR (LR-iPCR) method. Using these we have successfully isolated two highly homologous promoter regions of the dirigent like gene of respectively 1151 and 985 base pairs. In silico analyses confirmed the presence of various transcription motifs, including a TATA-box. However, experimental verification is needed to fully assess the functionality of these promoter regions. Verifying the activity of the isolated promoters through transient expression analysis proved to be problematic because of their highly mature culm specificity. Both constructs are therefore being used to obtain stable transformants in which promoter activity can be evaluated in mature internodal tissues.

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