PP4 in Arabidopsis
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This project will aim to unveil the subcellular localization of PP4-2 and two of its believed regulators; PP4R2L and PP4R3L/PSY2L. To achieve this, two fusion-proteins were prepared from each of the genes to be examined. This was done by using molecular cloning. One fusion-protein was designed to carry the EYFP tag on N-terminus and the other fusion-protein carried the EYFP tag on the C-terminus. Molecular cloning was not successful for the PSY2L gene, possibly due to its large size. The two PP4-2 fusion-proteins; PP4-2-EYFP and EYFP-PP4-2, appear to be cytosolic with clusters of protein aggregation and the two PP4R2L fusion-proteins also displayed cytosolic localization. No aggregation was observed for the PP4R2L fusion proteins. This thesis also wishes to examine the effect of artificial micro RNA (amiRNA) on PP4-2. Two different amiRNAs were used each with two different vectors; the inducible pER10 vector and the constitutive pBA002 vector. The goal was to do expression studies, and observe the phenotypes of the different mutant plants. Expression studies were not done due to a lack of time. No significant difference in phenotype was observed for the different mutant plants. They did however display slower growth rate than that of the wild type. Finally, one more study was performed to observe the effect of disrupting the PSY2L gene and the PP4R2L gene. This was done by studying plants with a T-DNA insert at specific locations in regards to the gene (see table 1), then observing the resulting phenotype and studying expression of the disrupted gene. For the PSY2L gene, mutants with T-DNA inserts at two different locations were used (one with the insert in exon 3, and one with the insert downstream of the gene) and T-DNA insert at only one location was performed for the PP4R2L gene (insert in exon 7). Expression study was to be preformed on homozygous individuals. At the time of the expression study, homozygous plants were found solely for the PSY2L Salk 125872 mutant. The expression study was performed on a homozygous PSY2L Salk 125872 mutant as well as one heterozygous PSY2L Salk 048064 mutant. The homozygous PSY2L Salk 125872 mutant displayed reduced expression when compared to the wild type. The heterozygous PSY2L Salk 048064 mutant displayed an expression level close to that of the wild type. At a later time, after the expression study had taken place, homozygous individuals for the PP4R2L Salk 093041 mutants were also found. No expression analysis was done on the PP4R2L Salk 093041 mutants, due to time restrictions. All mutant plants (T-DNA mutants and amiRNA mutants) displayed a reduced growth rate, as well as being shorter and bearing fewer stems than what is commonly observed for the wild type. The time taken for them to produce seeds was also about one month longer than what is observed for the wild type.
Master's thesis in Biological chemistry