Browsing by Author "Fernhout, Jean-Jacque"
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- ItemIsolation and characterisation of genes involved in carbon and chlorophyll metabolism in Saccharum species hybrids(Stellenbosch : Stellenbosch University, 2015-04) Fernhout, Jean-Jacque; Van der Vyver, Christell; Lloyd, James Richard; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB).ENGLISH ABSTRACT: Sugarcane is a tropical perennial grass species belonging to the Poaceae (true grasses) family. Mature sugarcane is comprised mostly of sugarcane stalks, which accumulate high amounts of sucrose, a fact that has led to its wide cultivation of sugarcane for sucrose production. Sugar yields from sugarcane have been improved in the past by either creating transgenic sugarcane or through using traditional breeding methods. Increasing sugar yields in sugarcane is still of interest and new cisgenic strategies are being considered to alleviate consumer concerns over transgenic plants. This thesis consists of two parts. The first was aimed at understanding the relation between trehalose-6-phosphate (T6P) synthesis and sucrose accumulation in sugarcane. In this study the E. coli genes involved in trehalose synthesis, otsA and otsB, were overexpressed in sugarcane in order to observe their effects on soluble sugar accumulation. Nine otsA and two otsB overexpressing lines were created, confirmed by gDNA insertion PCRs, sq-RT-PCR and immuno detection of encoded enzymes. Preliminary measurements of soluble sugars showed that four out of the nine otsA lines had significantly decreased and one line significantly increased sucrose concentrations. Correlating sq-RT-PCR results with soluble sugar measurements suggest that trehalose-6-phosphate synthase (TPS) expression affects sucrose levels in sugarcane, but further research of TPS activity is required before a conclusion can be reached. Further analysis of mature cane material in regard to relevant enzyme levels, carbohydrate levels and gene expression should contribute to more conclusive results. Three novel sugarcane TPS encoding sequences were isolated and proven to be functional through complementation of the growth defect in tps1Δ yeast grown on glucose as a carbon source. Sugarcane TPS isoforms named SoTPSa, SoTPSb and SoTPSc, were isolated by successful application of 5‟ RACE alongside standard PCR using primers based on other monocotyledonous TPS sequences. The encoded SoTPSa contains a 25 amino acid insertion within the partial TPP domain. The encoded SoTPSc contains a 126 amino acid long N terminal truncation, which removes one of the thirteen amino acids found within the active site of the TPS domain. Future characterization of the encoded enzymes will determine the effects of these modifications on TPS activity. The second part of this thesis describes initial efforts made in attempting to develop a cisgenic in vitro selectable marker system for sugarcane, S. officinarum callus, which uses a diphenylether type (DPE) herbicide as a selection agent and a sugarcane protoporphyrinogen oxidase (PPO) gene as a selection marker. Firstly the plastid targeted PPO from tobacco (NtPPO-1) was isolated and mutagenized, to mimic the double mutated Arabidopsis PPO, used by Li et al., (2003) in maize. However, sugarcane calli transformed with the double mutated NtPPO-1 and grown on media containing fomesafen herbicide, were incapable of regenerating. Future efforts will utilize a N-terminal sequence that is targeted to the plastid organelle, so as to ensure translocation of the enzyme to that subcellular location. Also, random mutations were induced in the NtPPO-1 gene to screen for mutations that confer DPE herbicide resistance, however this work is currently on hold until a heme deficient E. coli can be obtained. Secondly, attempts were made to isolate a putative sugarcane plastid targeted PPO gene, so as to eventually use this in developing a cisgenic strategy. 5‟ RACE was successful in revealing additional nucleotide sequence adding 1006 bp to the already known partial sugarcane PPO sequence. However the fragment isolated was still a partial sequence.