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Κωδικός Πόρου 000460380
Τίτλος Identification and functional characterization of RNA Silencing key-genes in the model pennate diatom species Phaeodactylum tricornutum
Άλλος τίτλος Ανεύρεση και λειτουργική ανάλυση γονιδίων του μηχανισμού της Γονιδιακής Σίγησης στο διάτομο, οργανισμό μοντέλο, Phaeodactylum tricornutum
Συγγραφέας Γρυπιώτη, Αιμιλία Σ
Σύμβουλος διατριβής Καλαντίδης, Κρίτων
Μέλος κριτικής επιτροπής Κωτούλα, Γεώργιο
Κοτζαμπάση, Κυριάκο
Σαρρή, Παναγιώτη
Καμπράνη, Σωτήρη
Πήττα, Παρασκευή
Falciatore, Angela
Περίληψη Gene silencing, also known as RNA interference (RNAi), is a conserved mechanism of regulation of gene expression mediated by small RNAs (sRNA), (Fire et al., 1998). Silencing of transgenes and endogenous genes following introduction of inverted repeats, antisense constructs and artificial miRNAs has been reported in diatom species, including the model species P. tricornutum (De Riso et al., 2009; Kaur and Spillane, 2015). The presence of an endogenous RNAi pathway has been suggested after comprehensive and combinatorial analyses of sRNAs, gene expression and DNA methylation in P. tricornutum (Veluchamy et al., 2013; Rogato et al., 2014). This RNAi pathway in P. tricornutum may play a role in the regulation of protein coding genes and TEs expression with possible consequences for the acclamatory response to nutrient limitation (Maumus et al., 2009). Homologues of the RNAi-key genes DICER (DCR), ARGONAUTE (AGO) and RNA-Dependent RNA polymerase (RDR) have been previously identified by in silico analysis (De Riso et al., 2009). However, the validation of their gene models, the characterization of their functions and the possible physiological role of RNAi in diatoms are still lacking. In this study, extensive in silico analysis of genomic and transcriptomic information available in P. tricornutum suggests the presence of a single PtDCR, PtAGO and PtRDR coding gene. Mining and phylogenetic analysis of DCR, AGO and RDR homologues in diatoms from all publically available to date sequence datasets suggest an unanticipated diversification of the RNAi pathway in these organisms. PtDCR/AGO/RDR cDNA were cloned and splicing isoforms of PtDCR and PtAGO were identified. Subcellular localization of PtDCR-/AGO-/RDR-YFP was investigated by confocal microscopy. Functional characterization of PtDCR and PtAGO was first attempted by heterologous expression in the yeast Saccharomyces cerevisiae and the plant Nicotiana bethamiana hosts. In a second step, CRISPR/Cas9-mediated mutagenesis approach, recently developed in P. tricornutum, was successfully harnessed to generate PtDCR-KO and PtAGO-KO (KnockOut) lines. Growth phenotype of PtDCR-KO lines were investigated under optimal and nitrate depleted culture conditions and during recovery from UVmediated stress. In parallel, mRNA and small RNAs whole transcriptome analyses were carried out. Culture experiments suggest that PtDCR may play a role in the response to nitrate starvation. Transcriptomic analysis revealed that both sRNA and mRNA transcriptomes were affected in PtDCR-KO line. At the global scale, sRNA size distribution was found to shift towards larger fragment size in PtDCR-KO line. In addition, the abundance of sRNA mapped to TEs was found dramatically reduced in PtDCR-KO mutant and a concomitant increase in mRNA abundance of some TEs was observed. Interestingly, PtDCR-KO sRNA transcriptome also presented changes in tRNA-derived sRNA populations, suggesting a possible role of DCR in their processing in diatoms. TE mobilization has been proposed to play a pivotal role in diatom species diversification and capacity for adaptation to various environments. Taken together, our results indicate that the single DCR encoding gene present in P. tricornutum plays a major role in the production of TE-derived sRNAs and possibly TE mobilization, with important consequences for diatom acclamatory response and evolution.
Φυσική περιγραφή xiii, [194] φύλλα : πίν., σχήμ., εικ. (μερ. εγχρ) ; 30 εκ.
Γλώσσα Αγγλικά
Θέμα Small RNAs
Διάτομα
Μικρά RNAs
Ημερομηνία έκδοσης 2023-11-15
Συλλογή   Σχολή/Τμήμα--Σχολή Θετικών και Τεχνολογικών Επιστημών--Τμήμα Βιολογίας--Διδακτορικές διατριβές
  Τύπος Εργασίας--Διδακτορικές διατριβές
Μόνιμη Σύνδεση https://elocus.lib.uoc.gr//dlib/9/8/a/metadata-dlib-1699960087-627785-17945.tkl Bookmark and Share
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