Year: 2007
Type: dissertation
Abstract: To date, genomic research into sucrose accumulation in sugarcane has focused on genes that are expressed in association with stalk development/maturation, with the aim of identifying key regulatory steps in sucrose metabolism. However, the identification of possible controlling points is complicated by the polyploid nature of the plant and these have therefore remained elusive. A close relative of sugarcane, sorghum, is diploid, has a small genome size and also accumulates sucrose in the stalk parenchyma. Here we provide a robust and reliable method for profiling multiple transcripts using sorghum as a model for sugarcane for studying carbohydrate metabolism. To determine the sensitivity of probe-target hybridisation, cDNA (the target) derived from young internodal tissue was probed with four randomly selected ESTs that were immobilised on nylon membranes in a macroarray format. The hybridisation signal increased as expected with increasing amounts of probe until the hybridisation signals reached maximum levels at 0.05 pmol probe. Using this as the probe concentration, 48 ESTs were arrayed in duplicate and hybridised with cDNA from pooled internodal tissues. Inter-array and intra-array variability was found to be low. Additionally, to test the reliability and sensitivity of the method, the target mRNA was spiked with an in vitro transcribed bacterial gene before cDNA synthesis. Variation in the spiked mRNA’s concentration was reliably detected. Under the conditions described here the technology is reproducible, robust and can reliably detect as little as two-fold changes in the levels of a specific transcript.
Author: Gordon Sandile Ndimande
Language: English
Subfield: Plant Science
Domain: Life Sciences
SDG: Zero hunger
Open Access status: green
OpenAlex ID: W2166444714