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Regulace biosyntézy vysoce nenasycených mastných kyselin (LC-PUFA) u ryb
BLÁHOVÁ, Zuzana
The aim of the thesis was to explore the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) with special focus on its role in adaptation to new environmental conditions in fish. The impact of impaired LC-PUFA pathway on reproduction of zebrafish (Danio rerio) was studied by the modern reverse genetic approach CRISPR/Cas9. Throughout the Earth´s history, the living organisms evolved to its current form in response to natural climate change, a slow natural phenomenon that has lasted for thousands or millions of years. However, in recent less than 100 years, anthropogenic factors induced a rapid process of global warming. Nutritional quality, when defined as the value of the product for the consumer´s physical health, growth, development and reproduction, is at a risk. Long-chain polyunsaturated fatty acid (LC-PUFA) biomolecules are essential dietary components for virtually all animals and humans. However, LC-PUFAs provisioning by changing ecosystems is predicted to decline significantly. Animals and human are usually able to produce LC-PUFAs endogenously from shorter and less unsaturated C18 fatty acid precursors obtained from the diet. However, the yield of LC-PUFA biomolecules biosynthesized is too low to meet physiological needs. Rather, LC-PUFA biosynthesis is considered to play a role as a compensational apparatus to maintain homeostasis under fluctuating environmental conditions. The first and rate-limiting step in LC-PUFA biosynthesis is catalysed by FADS2 enzyme. It has been demonstrated that fads2 was the key metabolic gene important for overcoming the nutritional constraints associated with colonisations of nutritionally pure water environments after glacial retreat. In human population, fads2 showed strong signatures of selection as well as confirmed in Greenland Eskimos colonising polar regions. Chapter 2 of the thesis summarized the current knowledge and assessed fatty acid desaturase (FADS2) structure-function properties in fish in the context of environmental adaptations and as a target for genetic engineering. The work highlighted the FADS2 potential to play roles fundamental to adaptation to novel environmental conditions and reviewed investigations to elucidate the evolutionary history of fish FADS2, explaining the remarkable plasticity of this enzyme in fish, depicted the importance of FADS2 in the context of dwindling LC-PUFAs supply and mentioned a few remedies in the form of genetic engineering to improve endogenous LC-PUFA biosynthesis. FADS2 was presented as a fascinating enzyme with far-reaching implications for environmental sustainability and playing roles in adaptations to novel environments. Previous research has provided substantial evidence of significant effects of dietarily obtained LC-PUFA levels and ?3: ?6 LC-PUFA balance on broodstock reproduction success (Sarih et al., 2020). However, the information on the impact of ablation of LC-PUFAs of endogenous origin was missing in fish and other anamniotic vertebrates as well. That fact prompted us to fill this gap of knowledge. Chapter 3 of this thesis investigated a direct functional link between fads2 gene and reproduction success in zebrafish (Danio rerio) by reverse genetic approach. We have investigated the functional link between FADS2 and reproduction success of zebrafish females by observing the direct impact of partial fads2 gene knock out on their egg batches fatty acyl methyl ester (FAMEs) profile. We observed the presence of an alternative ?8 desaturation pathway (elongation ? ?8 desaturation ? ?5 desaturation) besides the classical ?6 pathway (?6 desaturation ? elongation? ?5 desaturation) in zebrafish in vivo, which we explain as an active regulation of LC-PUFA biosynthesis under the depletion of functional fads2 gene alleles following the CRISPR/Cas9 genome targeting. We suggest that zebrafish bifunctional ?6/ ?5 desaturase is, in fact, the trifunctional ?8/?6/?5 desaturase.
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