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Chew, Shit Fun
Plant lipid metabolism pathways have been extensively studied as increasing plant oil yields is one important goal among researchers. However, the gene expression pattern of lipid metabolism regulating pathways especially in wound-induced callus of Arabidopsis thaliana is still unknown. The use of artificial microRNA retinoblastoma-related (amiRBR) strain allowed for the study of reduced RBR expression effects on lipid accumulation. Although the lipid contents in hormone-induced callus were possibly lower than leaves in wild-type (WT) A. thaliana, wound-induced callus was used to eliminate genetic interferences. Lipid content comparisons on 7-, 14- and 21-day old leaf and callus showed possibly higher lipid content in WT callus, although amiRBR callus contained possibly higher lipid content than its leaves at Day 7. Lipid metabolism genes undergoing cell-fate changes were studied. The mRNA expression of the lipid catabolism genes, acyl-CoA oxidase 1 and 2 (ACX1, ACX2) and multifunctional protein 2 (MFP2) were not significantly different in WT from Day 7 to Day 21. Sugar-dependent 1 (SDP1) increased significantly from Day 7 to Day 21 in WT and 3-ketoacyl-CoA thiolase 2 (KAT2) increased significantly from Day 7 to Day 14. ACX1, ACX2, MFP2 and SDP1 reached expression levels of 2 across the time-points in amiRBR callus with ACX1 expression significantly lower at Day 14 in amiRBR compared to WT, suggesting that cell death induced by RBR knockdown may have a negative effect on the expression of these genes. KAT2 expression increased from a fold change of 2 to 12 (Day 7 to Day 21) in amiRBR callus, though the difference was not significant. Compared to WT, transcript levels of long-chain acyl-CoA synthetase 6 and 7 were not different in amiRBR. Surprisingly, expression of diacylglycerol acyltransferase 2 was kept below fold change of 2 for both WT and amiRBR across Day 7 to Day 21 hence overexpression assay was performed. Diacylglycerol acyltransferase 1 was up-regulated in WT for all the time-points. Transcript levels of lipid transport genes acyl-CoA binding domain protein 1 and 3 and comatose were fluctuating between 0.8 to 1.5 folds, with no detectable difference between WT and amiRBR. This suggested that transport genes activities were uninterrupted across strains. Target gene expression cassettes of MFP2 and KAT2 were constructed using clustered regularly interspaced short palindromic repeats associated protein 9 with the successful targeted deletion of these genes in WT and amiRBR A. thaliana through Agrobacterium tumefacien-mediated transfection producing insertion-deletion mutation. Transcript levels of MFP2 and KAT2 mutants of both WT and amiRBR plants were below 1-fold change, indicating suppression of gene activities.
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