| dc.description.abstract | Diatoms, a group of unicellular photosynthetic microalgae, are of high ecological importance. They are responsible for about 20% of the world’s oxygen production. They have also been exploited in research due to their high potential for biofuel production, fatty acid extraction and in research as a model species for photosynthetic organisms in general. Both basic research and improvement of their favorable traits for commercial applications require methods for genome editing and regulation of gene expression. Many of these methods, including CRISPR/Cas9 to create knock-out strains and RNAi to downregulate gene expression have already been established in the model species Phaeodactylum tricornutum. Proteins from the chloroplast signal recognition particle (cpSRP) have been the focus of interest for the generation of mutant strains with increased photosynthetic productivity for industrial production. Knock-out mutants of cpSRP proteins in plants, green algae and diatoms have shown lower pigment levels and pale coloration, due to the decrease in light-harvesting complex proteins (LHCPs). Two ALBINO3 (ALB3) proteins, ALB3a and ALB3b, members of the cpSRP, have been targeted in P. tricornutum for the same purpose. Knock-out mutants of ALB3b showed similar phenotype as the rest of the cpSRP mutants, whereas no ALB3a knock-outs could be generated, suggesting it to be an essential gene. Thus, a method for downregulation of this protein was needed. The species P. tricornutum has been used in this project to develop a novel method for downregulation of target genes using an episome carrying CRISPR/Cas13 delivered by trans-kingdom conjugation. Cas13 has been shown to be more specific than RNAi, resulting in less off-target effects. Two target genes were chosen: ALB3a and ALB3b While creation of knock-down strains of ALB3a would be most valuable for further research, it was not known how much downregulation of the transcript was possible. Therefore, knock-down of ALB3b was performed in parallel due to its known phenotype of reduced pigment content in knock-out strains. As the experiment was the first attempt to develop this method in P. tricornutum, it served to consider possible improvements that could be applied, as well as the limitations of the method. It was possible to generate different degrees of downregulation in alb3a mutants, meaning that this method shows a strong potential as an alternative for gene downregulation in diatoms. The levels of downregulation obtained in alb3a mutants did not seem to be stable over a long period, due to the type of protein that was being targeted. In order to confirm that the Cas13 method for downregulation can be reliable for its use in protein characterization in diatoms, further improvements are needed. The results obtained relate to previous publications that tried to describe the Alb3 proteins and to elucidate their role in the integration of pigment-binding proteins into the thylakoid membrane. | |