Masters Degrees (Genetics)
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- ItemIdentification and characterisation of a drought-responsive galactinol synthase in wild rocket (Diplotaxis tenuifolia)(Stellenbosch : Stellenbosch University, 2023-12) Xonti, Alukhanyo; Peters, Shaun W.; Loedolff, Bianke; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are sucrosyl-galactosides that occur uniquely in the plant kingdom. The elucidated functional roles attributed to RFOs include serving as carbohydrate transporters, storage reserves and acting as protective agents against biotic and abiotic stress effects. Galactinol synthase (GolS; EC 2.4.1.123) is the key biocatalyst of the RFO biosynthetic pathway and initiates the synthesis of the precursor galactinol (Gol) with the transfer of a galactosyl moiety from UDP-galactose (UDP-Gal) to myo-inositol (Ino). RFO biosynthesis involves the sequential addition of activated galactosyl moieties from Gol to sucrose (Suc) with the enzymes raffinose synthase (RafS; EC 2.4.1.82) and stachyose synthase (StaS; EC 2.4.1.67) to produce raffinose (Raf) and stachyose (Sta) plus higher order RFOs respectively. The coupled transcriptional upregulation of GolS and subsequent RFO accumulation during water deficit stress has been well-studied in several plants; however, these functional dynamics have not been explored in wild rocket (Diplotaxis tenuifolia). The focus of the current study was to isolate and functionally characterise a putative DtGolS1 and investigate its transcriptional regulation and subsequent water-soluble carbohydrates (WSC) profile change in D. tenuifolia leaves under mild water deficit stress. With the heterologous expression of DtGolS1 in E. coli (DH5α), it was demonstrated that the extracts synthesized galactinol in vitro. The predicted DtGolS1 amino acid sequence exhibited GolS hallmarks from other plant species including the C-terminal APSAA pentapeptide and a serine phosphorylating site. Transcriptional analyses also indicated that DtGolS1 is sensitive to water deficit stress as transcript levels observed an upregulation for the most stressed rocket plants. Based on the current findings, the identification of a promising DtGolS1 candidate gene for sugar metabolic manipulation in improving water stress tolerance in wild rocket was revealed. In addition, owing to the prebiotic properties of RFOs, a greater understanding of RFO metabolism particularly in salad greens such as wild rocket could provide insight into biofortification strategies leading to the production of sustainable salad crops.
- ItemDoes lumichrome-enhanced plant growth reduce disease resistance?(Stellenbosch : Stellenbosch University, 2023-12) Henriques, Miguel Stephen Joel; Hills, Paul N. ; Moore, John P. ; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: The need for an increased food production has brought about interest in the study of plant growth-promoting rhizobacteria (PGPR) and their extracts, known to have positive effects on plant growth. One such substance is lumichrome (7,8 dimethylalloxazine), which was first identified as a growth promoting substance from the nitrogen-fixing soil bacterium, Sinorhizobium meliloti and has been shown in previous research to enhance plant growth through upregulated photosynthesis, cell cycle, and development genes, thereby increasing stomatal conductance, transpiration, photosynthesis, and biomass accumulation in the plants. However, downregulation of defence genes was also observed after lumichrome treatment in Arabidopsis thaliana wild type (wt) plants, which suggested an increase in their susceptibility to pathogen attack. Starchless Arabidopsis mutant plants (aps1) deficient in ADP-glucose pyrophosphorylase small subunit (APS1) were previously found to have similar levels of photosynthesis and enhanced growth to lumichrome-treated wt plants, even without being treated with lumichrome. This suggested that the loss-of-function APS1 gene (which was also downregulated by lumichrome in wt plants) contributed significantly to the enhanced growth seen in these mutants. The aim of this study was to treat Arabidopsis thaliana plants with lumichrome to determine the defence response that lumichrome has on plants. This was accomplished by treating Col-0 wt and adg1-1 plants (aps1 seeds were not available, so adg1-1 seeds, which have a mutation in the same gene, were used) with 5 nM lumichrome to observe the phenotypic and transcription effects of lumichrome on the growth of these plant lines. Physiological parameters such as rosette diameter, leaf number, and biomass, stomatal conductance, and transpiration rate were measured; while photosynthetic pigment and carbohydrate assays were conducted to observe differences caused by lumichrome treatment. RT-qPCR was used to assess the effects of lumichrome on the transcription of several lumichrome response-related marker genes. The necrotrophic pathogen Botrytis cinerea was then used to infect the lumichrome-treated plants. Fungal lesions were measured, and leaves were stained with DAB (3,3’-diaminobenzidine) and trypan blue to determine changes in the plants’ physiological response. RT-qPCR was conducted on infected leaves to determine transcriptomic reactions of lumichrome-treated plants to infection. No significant physiological differences were observed in wt or adg1-1 mutants following lumichrome treatment. However, RT-qPCR analysis revealed an upregulation of XTH9 which is involved in tissue division and expansion. Upon lumichrome treatment, downregulation was observed in psbTn, involved in photosynthesis; AGPase, involved in starch biosynthesis; CYCA1;1, involved in cell cycle progression; ACO1, involved in ethylene biosynthesis; PR1, involved in salicylic acid signalling; JAL34, involved in endoplasmic reticulum body production; and PDF1.2A, involved in jasmonic acid induction. DAB staining revealed that lumichrome treatment may have caused increased levels of hydrogen peroxide accumulation both in the uninfected leaves and directly around the lesions of infected plants, indicating increased levels of hypersensitive response in these plants. Trypan blue staining confirmed increased cell death in lumichrome- treated plants, likely caused by the hypersensitive response directly around the fungi. Analysis of the infected leaves by RT-qPCR revealed that lumichrome reduces the transcription of the JAL34, PDF1.2A, and PR1 defence genes, making plants more susceptible to necrotrophic pathogens.
- ItemDevelopment of genome editing of potato to repress cold-induced sweetening(Stellenbosch : Stellenbosch University, 2023-12) Schwegler, David Alexander; Lloyd, James; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: In South Africa, the potato (Solanum tuberosum L.) has become one of the country’s most significant food sources. Its tubers are an abundant source of dietary carbohydrates for human consumption and contain starch, a polymeric carbohydrate composed of amylose and amylopectin that also has numerous industrial uses. Potatoes are often stored at cold temperatures after harvesting, leading to starch being degraded to form the reducing sugars glucose and fructose. When heated to high temperatures, these sugars react with amino acids in the Maillard reaction to produce acrylamide that is neurotoxic and potentially carcinogenic. It is therefore desirable to produce plants with tubers that don’t produce reducing sugars when stored at cold temperatures. An enzyme that plays a critical role in starch degradation is α-glucan, water dikinase 1 (GWD1), and plants without GWD1 activity have demonstrated impaired starch degradation including in cold-stored potato tubers. Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 is a multipurpose technology for genetic engineering and provides new opportunities for developing novel plant characteristics. This system provides a novel method to mutate StGWD1 to prevent starch degradation. To accomplish this, single guide RNAs were designed to target regions of the gene before a key catalytic phosphohistidine site that is essential for enzyme activity. The guides were transcribed in vitro and complexed with Cas9 to assess cleavage of a target-containing sequence. The in vitro efficacy assay demonstrated that one of the guides successfully introduced a double-stranded break to the target sequence. Two strategies were used to try to mutate StGWD1. The first was a transgene-free method for gene editing that required successful protoplast isolation and regeneration. After successfully isolating viable potato protoplasts, micro-calli were generated from tissue culture of these cells. Unfortunately, regeneration did not proceed further and so a transgenic approach using Agrobacterium- mediated transformation was then used to transform leaf explants. This resulted in transgenic calli, although sequencing reactions and Tracking of Indels by Decomposition (TIDE) analysis revealed no editing at the target sequences.
- ItemCharacterising the epigenetic architecture of differential treatment outcomes in a South African first-episode schizophrenia cohort with emphasis on miRNA-mediated regulation(Stellenbosch : Stellenbosch University, 2023-12) Jooste, Sarel Christiaan; Mcgregor, Nathaniel Wade; Pearce, Brendon; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Approximately 24 million individuals of the global population are affected by schizophrenia. Strikingly, antipsychotic treatment remains effective in only half of these individuals. This is a product of the complex multifactorial nature of the disorder and even more so its treatment. It has become a necessity to utilize the genetic and neurological underpinnings of the disorder, to elucidate and potentially improve on actionable clinical treatments, such as an improved susceptibility risk estimates and point-of-care diagnosis detection devices. Genome-Wide Association studies provide remarkable insights into the heritable component of neuropsychiatric disorders, although they still only explains a part of the presentation of the disorder. Variations in neuronal structure, as well as other environmental and epigenetic influences critically contribute to the interplay between the aetiology and treatment of schizophrenia. The hyper-focus on the direct genomic structure alone is perhaps due for re-evaluation into potential biomarkers that may play a more regulatory role, such as the cascade of epigenetic effects of miRNA. Biomarkers, briefly defined as a DNA sequence associated to the susceptibility of a biological risk, may also be directly measurable and may lead to greater insights into the missing heritability of the pharmacogenetics of antipsychotic medication. This study primarily focused on miRNAs to identify potential biomarkers for neuropsychiatric disorders and specifically differential antipsychotic treatment responses. A special focus was placed on hsa-miR-548al, a miRNA which has previously been implicated in the pathophysiology of schizophrenia and treatment thereof. For the purposes of this study, a particular single nucleotide polymorphism, rs515924, in the seed sequence of the focal miRNA was specifically investigated. The study utilizes 103 first-episode schizophrenia patients treated with long-acting injectable flupentixol decanoate, as well as mixed- ancestry genome wide association studies data from schizophrenic individuals. Candidate genes that are influenced by the target miRNA were identified. Finally, mixed-effects linear regression and logistic regression association analyses were performed to assess the statistical significance of the miRNA’s impact on differential treatment responses in schizophrenia. The results shows clear trends towards nominal significance (p-value < 0.05) for the miRNA SNP hsa-miR-548al rs515924, as well as for candidate SNPs found in genes predicted to be affected by hsa-miR-548al to be associated with differential antipsychotic treatment responses. These associations were found to be modified when including the severity of childhood trauma as an interacting variable. The dysregulation of hsa-miR- 548al or its target genes may impact molecular pathways related to differential treatment outcomes, such as the neuronal developmental pathways and immune pathways, highlighting the importance of miRNA-mediated gene regulation. The findings suggest that rs515924 could serve as a predictive marker for treatment response and inform tailored treatment approaches. This research contributes to filling the knowledge gap in understanding the complex interplay between genetic and epigenetic variations and clinical phenotypes in differential antipsychotic treatment response.
- ItemAnalysis of starch metabolism in South African pigeon pea (Cajanus cajan) varieties(Stellenbosch : Stellenbosch University, 2023-03) Kulu, Nokwanda; Lloyd, James R. ; Peters, Shaun Wayne; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.ENGLISH ABSTRACT: Starch is a major storage polyglucan in plants that is composed of two fractions, amylose and amylopectin. The biosynthesis and degradation pathways of starch are well documented, with phosphoglucomutase (PGM) and ADP-glucose pyrophosphorylase (AGPase) catalysing the first two steps in its biosynthesis. This project examined starch in five pigeon pea (Cajanus cajan) varieties: uDhali, SEFA, Nondolo, Lari and India by measuring both total and resistant starches in the seeds and leaves, activities of PGM and AGPase as well as expression of the genes encoding these enzymes. The findings demonstrated that the seeds from these South African pigeon pea varieties are rich in starch, containing an average of 47% starch on a dry weight basis; however, one variety (SEFA) contained only 0.3% starch. The starch in the high- starch varieties contained a minimum of 50% resistant starch, with the India variety reaching 70%. Assessment of soluble sugars in seeds revealed sucrose to be the only sugar present in abundance in all varieties while amounts of galacto-oligosaccharides were low in all seeds. Starch in leaves was observed to be 10 fold less than that found in seeds and the amount of resistant starch in leaves was less than 2 mg/g fresh weight (7.6% of the total). The AGPase gDNA nucleotide sequence from one variety was identical to an already sequenced pigeon pea variety, whereas amplification PGM gDNA was unsuccessful. Amplification of coding sequences (CDSs) for both AGPase and PGM were also identified to be the same as the already sequenced AGPase and PGM genes from the pigeon pea genome resource database. Gene expression for both genes varied throughout a 24 h period and was at its peak during the day (light period). Activities of both AGPase and PGM were determined in seeds from all varieties whereas the AGPase enzyme activity was the same in leaves throughout the day while PGM activity varied between the day (light) and night (dark).