Resumo: O gênero Zaprionus tem sido eleito como um bom modelo biológico para estudos genéticocomparativos com as espécies do subgrupo melanogaster do gênero Drosophila, embora seu posicionamento filogenético dentro da família Drosophilidae ainda seja controverso. Na presente Tese foi investigada a presença de 10 elementos de transposição (TEs) em Zaprionus indianus e Drosophila malerkotliana, bem como a distribuição, a atividade transcricional e as relações evolutivas de três retrotransposons (copia, gypsy e micropia) em sete espécies do gênero Zaprionus. Para isso, foram empregadas as técnicas de Dot blot, PCR, RT-PCR e seqüenciamento. As seqüências obtidas foram comparadas às dos respectivos elementos das demais espécies de drosofilídeos disponíveis nas bases de dados genômicas. Os resultados indicam que Z. indianus e D. malerkotliana apresentam em seus genomas todos os TEs de D. melanogaster investigados. O retrotransposon copia foi seqüenciado e está transcricionalmente ativo nas sete espécies do gênero Zaprionus e constitui uma nova subfamília relacionada aos elementos do subgrupo melanogaster, que foi denominada subfamília GBFDouble-gap. Por outro lado, os retrotransposons gypsy e micropia foram identificados nas espécies do subgênero Zaprionus, onde também estão transcricionalmente ativos, e pertencem às subfamílias já descritas para as espécies do subgrupo melanogaster. As análises evolutivas sugeriram que esses três retrotransposons devem ter participado de eventos de transferência horizontal com as espécies do subgrupo melanogaster e com pelo menos um doador desconhecido, no caso do retrotransposon micropia. Além disso, o cálculo dos tempos de divergência dos elementos sugere que eles passaram por ondas de transferências horizontais, mais antigas para o retrotransposon copia, e mais recentes para gypsy e micropia. Esses resultados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Zaprionus genus has been elected as a good biological model for comparative analyses with the melanogaster subgroup of Drosophila genus, though its phylogenetic positioning within the Drosophilidae family is still controversial. This study aiming at investigating the occurrence of 10 transposable elements (TEs) in Zaprionus indianus and Drosophila malerkotliana species, as well the distribution, transcriptional activity and evolutionary relationships of three retrotransposons (copy, gypsy and micropia) in seven species of Zaprionus genus. To do so, Dot blot, PCR, RT-PCR and sequencing methods were employed. The Zaprionus sequences obtained were compared with the drosophilid sequences available in genomic databases. The results indicated that Z. indianus and D. malerkotliana harbor all D. melanogaster TEs investigated. The copia retrotransposon is present and transcriptionally active in seven species of the Zaprionus genus and represents a new subfamily related to that of the melanogaster subgroup, named as GBFDouble-gap subfamily. Additionally, gypsy and micropia retrotransposons were identified in the Zaprionus species subgenus, which are transcriptionally active and belong to the melanogaster subgroup subfamilies. The evolutionary analysis showed the three retrotransposons could have been involved in horizontal transfer events with species of the melanogaster subgroup for the three retrotransposons and at least one unknown donor regarding to micropia retrotransposon. Moreover, the time of divergence seems to indicate that the retrotransposons experienced horizontal transfer waves, the oldest involving the copia element followed by the gypsy and micropia retrotransposons in more recent times. These results suggest that the horizontal transfer phenomenon has happened repeatedly during the Zaprionus genus and melanogaster subgroup evolution in the Afrotropical region. / Orientador: Cláudia Márcia Aparecida Carareto / Coorientador: Marie Anne Van Sluys / Banca: Hermione Elly Melara de Campos Bicudo / Banca: Maria Magdalena Rossi / Banca: Galina Ananina / Banca: Elgion da Silva Loreto / Doutor

Made available in DSpace on 2015-03-26T13:51:59Z (GMT). No. of bitstreams: 1 texto completo.pdf: 536332 bytes, checksum: 5951891794c17e210fb52fda2d6fa297 (MD5) Previous issue date: 2013-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Devido à ausência de estudo de caracterização da variabilidade genética das populações de Pseudocercospora fijiensis recentemente introduzidas no Brasil, o objetivo desse trabalho foi avaliar a adequabilidade do marcador IRAP para estudar variações genéticas entre indivíduos, bem como determinar a estrutura genética da população brasileira de P. fijiensis com base no fingerprinting gerado por amplificação de polimorfismo entre retrotransposons (IRAP). Um total de 22 locos foi amplificado, sendo 77.3 % polimórficos. A análise de agrupamento revelou dois principais grupos no Brasil. A diversidade gênica (HE) foi de 0.22 e pela análise de variância molecular verificou-se que a maior variabilidade genética está dentro das populações. A Análise Discriminante de Componente Principal (DAPC) revelou que não há nenhuma estruturação relacionada com as origens geográficas e cultiva hospedeiro. O sistema de marcador baseado em retrotransposon IRAP é ferramenta apropriada para estudar a variabilidade genética em P. fijiensis. / Due to the lack of characterization study of genetic variability in populations of Pseudocercospora fijiensis recently introduced in Brazil, the objective of this study was to evaluate the suitability of IRAP marker for studying genetic variations between individuals, and to determine the genetic structure of the population of P. fijiensis based on fingerprinting generated by inter-retrotransposons amplified polymorphism (IRAP). A total of 22 loci were amplified and 77.3% showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (HE) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The Discriminant Analysis of Principal Components (DAPC) revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.

Afin d'étudier la formation de l'hétérochromatine dans l’embryon préimplantatoire de souris, je me suis concentrée sur deux régions génétiques différentes - répétitions péricentriques et L1 éléments transposables - dans le but notamment de découvrir les mécanismes qui conduisent à la répression et le rôle distinct qu’ils peuvent jouer pendant le processus de développement et la division cellulaire. Mes expériences montrent que l’organisation spatiale spécifique des domaines péricentriques est essentielle pour leur répression ainsi que pour leur organisation correcte. De plus, mes résultats suggèrent que les défauts d’organisation de l’hétérochromatine conduisent à des défauts de division cellulaire et de prolifération. La seconde partie de ma thèse montre que la réglementation stricte de L1 éléments transposables est nécessaire pour le développement préimplantatoire d'embryons de souris. En outre, représente la première tentative pour élucider la biologie des éléments L1 dans l’embryon précoce de souris par l’utilisation de modificateurs de transcription ciblés spécifiquement. / To study the formation of heterochromatin in mouse preimplantation embryo, I focused on two different genetic regions – pericentric repeats and L1 transposable elements - in order to investigate the mechanisms that lead to their repression and the distinct role that these regions can play during the process of development and cell division. My experiments show that the specific spatial organization of pericentric domains is essential for their repression and for their correct organization. Moreover, my findings suggest that defects in organization of heterochromatin lead to improper cell division and proliferation. The second part of my thesis shows that the tight regulation of L1 transposable elements is required for the preimplantation development of mouse embryos. Additionally, it is the first attempt to elucidate the biology of L1 elements in the early mouse embryo through the use of targeted transcription modifiers.

Retrotransposons are major components of plant genomes influencing their genome size, organization and evolution. In the frame of this work, retrotransposons of the Beta vulgaris genome have been identified by molecular methods and whole genome bioinformatics approaches. Neither belonging to the rosids nor asterids, B. vulgaris (cultivated beet including sugar beet, beet root and mangold) is taxonomically placed at a key position at the root of the core eudicots, and considerably different from traditional plant model species such as thale cress or rice. Its genome has been sequenced, and annotation is under way. In order to compare different evolutionary lineages of B. vulgaris retrotransposons, long terminal repeat (LTR) and non-LTR retrotransposon family have been analyzed in detail. Full-length members have been isolated and characterized by bioinformatics, Southern and fluorescent in situ hybridization. Hallmarks of the LTR retrotransposon family Cotzilla are an additional env-like open reading frame (ORF), homogeneity of the members and the very high abundance. Most family members are evolutionarily young, and have most likely been created during recent bursts of amplification during species radiation. In contrast, the non-LTR retrotransposon family BNR has fewer copies and is much more diverged. Although the BNR ORF2 resembles previously analyzed long interspersed nuclear elements (LINEs) of the L1 clade, its ORF1 sequence differs strongly. It lacks the zinc finger domain described for plant LINEs, but contains instead an RNA recognition motif (RRM) likely to have an RNA-binding function. Database searches revealed the presence of similar LINE families in higher plant genomes such as poplar, lotus and soybean. Comparing their reverse transcriptase regions with other retrotransposons, these BNR-like LINEs form a separate group of L1 LINEs designated as BNR subclade. Availability of the B. vulgaris genome sequence allowed retrotransposon analyses on a genome-wide scale. A Hidden Markov Model-based detection algorithm has been developed in order to retrieve retrotransposon information directly from the database. Nearly 6000 B. vulgaris reverse transcriptase sequences have been isolated and classified into LTR retrotransposons of the Ty3-gypsy and Ty1-copia type, and non-LTR retrotransposons of the LINE type. As a result, a comprehensive overview of the retrotransposon spectrum of the B. vulgaris genome has been generated. Since plant LINEs have been only rarely investigated, the B. vulgaris LINE composition was studied in detail. Out of 28 described LINE clades, only members of the L1 and RTE clades have been identified. Based on a minimal shared sequence identity of 60 %, they form at least 17 L1 families and one RTE family. Full-length members of all investigated L1 families have been analyzed regarding their sequence, structure and diversity. In order to transfer the algorithm tested in B. vulgaris to other angiosperm genomes, twelve additional plant genomes have been queried for LINE reverse transcriptases. Key finding is the presence of only two LINE clades (L1 and RTE) in the analyzed genomes of higher plants. Whereas plant L1 LINEs are highly diverse and form at least seven subclades with members across species borders, RTE LINEs are extremely homogenized and constitute most likely only a single family per genome. In summary, this work’s results help to gain an understanding of the different strategies of retrotransposon evolution in plants, whereas the generated data directly contributes to the B. vulgaris genome annotation project. / Retrotransposons sind eine wesentliche Komponente von Pflanzengenomen, die sowohl die Größe und Organisation als auch die Evolution dieser Genome wesentlich beeinflussen können. Im Rahmen dieser Arbeit wurden verschiedene Gruppen von Retrotransposons des Beta vulgaris Genoms mittels molekularer und bioinformatischer Methoden identifiziert. Innerhalb der dikotyledonen Blütenpflanzen gehört B. vulgaris (kultivierte Rübe einschließlich Zuckerrübe, Roter Beete und Mangold) weder zu den Rosiden noch zu den Asteriden, sondern nimmt eine Schlüsselposition innerhalb der Kerneudikotyledonen ein. Somit zeigt das Rübengenom wesentliche Unterschiede zu traditionellen Modellpflanzen wie Arabidopsis thaliana oder Oryza sativa. Das Genom ist bereits sequenziert, die Annotation jedoch noch nicht abgeschlossen. Um verschiedene evolutionäre Linien von B. vulgaris Retrotransposons vergleichend zu untersuchen wurden insbesondere Long Terminal Repeat (LTR)- und Non-LTR-Retrotransposon-Familien detailliert analysiert. Vollständige Mitglieder wurden isoliert und mittels bioinformatischer Methoden, Southern- und Fluoreszenz-in situ-Hybridisierung untersucht. Die LTR-Retrotransposon-Familie Cotzilla ist durch einen zusätzlichen env-ähnlichen offenen Leserahmen (ORF), Homogenität ihrer Mitglieder und eine hohe Abundanz gekennzeichnet. Die meisten Cotzilla-Kopien sind evolutionär jung und wurden wahrscheinlich innerhalb eines kurzen Zeitraumes während der Artentstehung stark amplifiziert. Im Gegensatz zur Cotzilla-Familie besitzt die Non-LTR-Retrotransposon-Familie BNR weniger Kopien und ist wesentlich divergierter. Während der BNR-spezifische ORF2 starke Ähnlichkeiten zu anderen pflanzlichen Long Interspersed Nuclear Elements (LINEs) der L1-Klade aufweist, unterscheidet sich der BNR ORF1 von diesen sehr stark. Im Gegensatz zu bereits beschrieben pflanzlichen LINEs kodiert er kein Zinkfingermotiv, sondern substituiert dieses durch ein RNA-Erkennungsmotiv (RRM). Durch Datenbanksuche konnten BNR-ähnliche LINEs in den Genomen höherer Pflanzen wie Soja, Lotus und Pappel identifiziert werden. Ein Vergleich der entsprechenden Reversen Transkriptasen (RT) mit den RTs anderer Retrotransposons zeigt, dass die BNR-ähnlichen LINEs eine separate Gruppe innerhalb der L1 LINEs bilden. Diese wurde daher als BNR-Subklade definiert. Die Untersuchung von Retrotransposons auf Genomebene wurde durch die B. vulgaris Genomsequenz ermöglicht. Um Retrotransposon-Informationen direkt aus dem Genom zu extrahieren, wurde ein Hidden Markov Modell (HMM)-basierter Detektions-algorithmus entwickelt. Annähernd 6000 B. vulgaris Reverse Transkriptase-Sequenzen konnten identifiziert und in LTR-Retrotransposons des Ty3-gypsy- beziehungsweise des Ty1-copia-Typs und in Non-LTR-Retrotransposons des LINE-Typs klassifiziert werden. Somit wurde ein umfassender Überblick über die Bandbreite der B. vulgaris Retrotransposons arhalten. Da pflanzliche LINEs bisher nur wenig erforscht sind, wurde die B. vulgaris LINE Zusammensetzung genauer untersucht. Von 28 beschriebenen LINE-Kladen konnten nur Mitglieder der L1- und der RTE-Klade identifiziert werden. Basierend auf einer Identität von mindestens 60 % bilden die Sequenzen 17 L1 Familien und eine RTE Familie. Vollständige Mitglieder aller L1 Familien wurden hinsichtlich ihrer Sequenz, Struktur und Diversität analysiert. Um den in B. vulgaris getesteten HMM-basierten Algorithmus auf andere Angiospermengenome zu übertragen, wurden zwölf weitere Pflanzengenome auf das Vorhandensein von LINE-spezifischen Reversen Transkriptasen untersucht. Wesentlichstes Ergebnis ist der Nachweis von nur zwei LINE-Kladen (L1 und RTE) in höheren Pflanzen. Während pflanzliche L1 LINEs hochgradig divers sind und über Artgrenzen hinaus mindestens sieben Subkladen mit Vertretern verschiedener Pflanzen bilden, sind RTE LINEs extrem homogenisiert und stellen höchstwahrscheinlich nur eine einzelne Familie pro Genom einer Art dar. Zusammenfassend ermöglichen die Ergebnisse dieser Arbeit eine Erweiterung des Verständnisses der unterschiedlichen Evolutionsstrategien von Retrotransposons in Pflanzen. Zusätzlich tragen die gewonnen Daten zur Annotation des B. vulgaris Genoms bei.

La méthylation de l’ADN et les modifications post-traductionnelles des histones sont desmodifications épigénétiques qui interviennent dans la régulation des éléments transposables(ET) chez de nombreuses espèces. La proportion des ET dans les génomes varie selon lesespèces considérées et pose la question des mécanismes de régulation de ces ET. Au sein del’espèce Drosophila simulans, les populations naturelles présentent un polymorphisme uniquedans le nombre de copies des ET, ce qui en fait un excellent modèle pour étudier cettequestion. L’étude de la méthylation d’ADN et des modifications post-traductionnelles deshistones associées au rétrotransposon à LTR tirant dans la lignée germinale des populationsnaturelles a permis de montrer l’influence d’une copie d’ET sur la structure de la chromatineau site d’insertion. Dans un second volet, nous avons cherché à caractériser la méthylation del’ADN chez la drosophile, chez laquelle la fonction est encore mal connue. Nous avons, pardes approches spécifiques et globales, mesuré l’abondance de cette marque épigénétique chezla drosophile. Nous concluons que les taux de méthylation de l’ADN sont très faibles maisvariables entre espèces. Notre travail n’a pas permis de mettre en évidence un rôle de laméthylation de l’ADN dans le contrôle des ET, toutefois, nous ne pouvons pas exclure cesystème de régulation. / Epigenetic modifications such as DNA methylation and post-translational histonemodifications are involved in transposable elements (TE) silencing in many species. Theirrelative abundance in genomes ask the question of differences in regulation mecanismbetween species. Within the Drosophila simulans species, natural populations exibits a uniquepolymorphism in TE copy number, providing a powerfull tool for the analysis of TEregulation in population from the same specie. We analyzed DNA methylation and posttranslationalhistone modifications associated with the LTR retrotransposon tirant in thegermline of natural populations and report the influence of this element on chromatinestructure. DNA methylation is a wide-conserved epigenetic modification involved in generegulation and TE silencing but its function in drosophila remains missunderstood. Usingdifferent methods, we determined the abundance of methylated cytosines in drosophila, andshowed that methylation level are low and variable between species. Our results show lowevidence for a TE regulation system involving DNA methylation but this cannot be so farexcluded.

Short interspersed nuclear elements (SINEs) are small non-autonomous and heterogeneous retrotransposons, widespread in animals and plants and usually differentially propagated in related species resulting in genome-specific copy numbers. Within the monocots, the Poaceae (sweet grasses) is the largest and economically most important plant family. The distribution of 24 Poaceae SINE (PoaS) families, five of which showing a subfamily structure, was analyzed in five important cereals (Oryza sativa, Triticum aestivum, Hordeum vulgare, Sorghum bicolor, Zea mays), the energy crop Panicum virgatum and the model grass Brachypodium distachyon. The comparative investigation of SINE abundance and sequence diversity within Poaceae species provides insights into their species‐specific diversification and amplification. The PoaS families and subfamilies fall into two length and structural categories: simple SINEs of up to 180 bp and dimeric SINEs larger than 240 bp. Of 24 PoaS families, 20 are structurally related across species, in particular either in their 5′ or 3′ regions. Hence, reshuffling between SINEs, likely caused by nested insertions of full-lengh and truncated copies, is an important evolutionary mechanism of SINE formation. Most striking, the recently evolved homodimeric SINE family PoaS‐XIV occurs exclusively in wheat (T. aestivum) and consists of two tandemly arranged PoaS‐X.1 copies. Exemplary for deciduous tree species, the evolutionary history of SINE populations was examined in six Salicaceae genomes (Populus deltoides, Populus euphratica, Populus tremula, Populus tremuloides, Populus trichocarpa, Salix purpurea). Four of eleven Salicaceae SINE (SaliS) families exhibit a subfamily organization. The SaliS families consist of two groups, differing in their phylogenetic distribution pattern, sequence similarity and 3’ end structure. These groups probably emerged at different evolutionary periods of time: during the ‘salicoid duplication’ (~ 65 million years ago) in the Salix-Populus progenitor, and during the separation of the genus Salix (~ 45 - 65 million years ago), respectively. Similar to the PoaS families, the majority of the 20 SaliS families and subfamilies share regions of sequence similarity, providing evidence for SINE emergence by reshuffling. Furthermore, they also contain an evolutionarily young dimeric SINE family (SaliS-V), amplified only in two poplar genomes. The special feature of the Salicaceae SINEs is the contrast of the conservation of 5’ start motifs across species and SINE families compared to the high variability of 3’ ends within the SINE families, differing in sequence and length, presumably resulting from mutations in the poly(A) tail as a possible route for SINE elongation. Periods of increased transpositional activity promote the dissemination of novel 3’ ends. Thereby, evolutionarily older motifs are displaced leading to various 3’ end subpopulations within the SaliS families. Opposed to the PoaS families with a largely equal ratio of poly(A) to poly(T) tail SINEs, the SaliS families are exclusively terminated by adenine stretches. Among retrotransposon-based markers, SINEs are highly suitable for the development of molecular markers due to their unidirectional insertion and random distribution mainly in euchromatic genome regions, together with an easy and fast detection of the heterogeneous SINE families. As a prerequisite for the development of SINE-derived inter-SINE amplified polymorphism (ISAP) markers, 13 novel Theaceae SINE families (TheaS-I - TheaS-VII, TheaS-VIII.1 and TheaS-VIII.2, TheaS-IX - TheaS-XIII) were identified in the angiosperm tree species Camellia japonica. Moreover, six Pinaceae SINE families (PinS-I.1 and PinS-I.2, PinS-II – PinS-VI) were detected in the gymnosperm species Larix decidua. Compared to the SaliS and PoaS families, structural relationships are less frequent within the TheaS families and absent in the PinS families. The ISAP analysis revealed the genetic identity of Europe’s oldest historical camellia (C. japonica) trees indicating their vegetative propagation from the same ancestor specimen, which was probably the first living camellia on European ground introduced to England within the 18th century. Historical sources locate the native origin of this ancestral camellia specimen either in the Chinese province Yunnan or at the Japanese Gotō Islands. Comparative ISAPs showed no accordance to the Gotō camellia sample pool and appropriate Chinese reference samples were not available. However, the initial experiments demonstrated the potential of ISAP to resolve variations among natural populations. The ISAP application on angiosperm trees also concerned fast growing Populus clones grown in short rotation coppice plantations for energy production. The species-specific P. tremula ISAP primers might also be applied for the discrimination of hybrid poplar clones involving P. tremuloides genome portions, since SINEs of these two species are highly related. However, due to lineage-specific SINE evolution during speciation, cross-species applications are generally only successful to limited extent. The analysis of poplar hybrids composed of P. maximowiczii with either P. trichocarpa or P. nigra based on P. tremula ISAP primers showed a strongly reduced resolution. In forestry, hybrid larch (e.g. Larix × eurolepis) genotypes have to be selected from the offspring of Japanese (Larix kaempferi) and European larch (Larix decidua) crosses, as they exhibit superior growth rates compared to the parental species. Initial ISAP-based examinations of European larch genotypes provided less polymorphic banding patterns, probably resulting from general high levels of synteny and collinearities reported for gymnosperm species. Hence, the ISAP was combined with the AFLP technique to the novel marker system inter-SINE-restriction site amplified polymorphism (ISRAP). The amplicons originating from genomic regions between SINEs and EcoRI cleavage sites were visualized with the sensitive capillary gel electrophoresis. The ISRAP assays, based on EcoRI adapter primers combined with two different SINE-derived primers, resulted in a sufficient number of polymorphic peaks to distinguish the L. decidua genotypes investigated. Compared to ISAPs, the ISRAP approach provides the required resolution to differentiate highly similar larch genotypes.

Made available in DSpace on 2014-08-20T14:06:12Z (GMT). No. of bitstreams: 1 tese_taciane_finatto.pdf: 5834731 bytes, checksum: e10f781234d54582cc17a9b8dff16c53 (MD5) Previous issue date: 2012-02-27 / Iron toxicity in plants is associated with the presence of large concentrations of reduced iron (Fe2+) in the soil solution, which occurs in flooded soils and affects rice plants grown under this condition. Symptoms of iron toxicity involve oxidative stress in leaves, as a response to excessive Fe2+ absorption by the roots. The responses of plants to stress conditions include stimulus perception, signal transduction and gene transcription activation. Besides gene expression, LTR (Long Terminal Repeat) retrotransposons represent ca. 22% of the rice genome, they can be transcriptionally activated under stress, and they can alter the expression of adjacent genes (e.g. due to alterations in chromatin structure). This study aimed to identify differentially expressed genes and LTR retrotransposons in leaves of 18-day-old rice seedlings (Oryza sativa ssp. japonica cv. Nipponbare) after four days of iron excess exposure. They were identified a differential expression of genes and LTR retrotransposons in rice exposed to iron excess using a microarray approach. Total RNA was extracted from leaves of 18-day-old rice seedlings (Oryza sativa L. ssp japonica cv. Nipponbare) after four days of cultivation in nutrient solution with iron excess (7 mM of FeSO47H2O) and in a control solution. The hybridization was performed with cDNA and rice transposome array v. 2.0 microarray (Roche/NimbleGen technology, an improvement of v.1.0, Picault et al., 2009). Data from gene expression was analyzed by the Bayesian t-test with BH adjustment method. Gene annotation, gene ontology, and LTR retrotransposon identification were performed at RAP-DB (Rice Annotation Project Database, build 5), and microarray results were validated by RT-qPCR. Considering log2 FC (log2-fold-change) ≤ -1 as underexpression and ≥ 1 as overexpression (p-values ≤ 0.05), 44 down-regulated and 1,572 up-regulated genes with described function were identified. Down-regulated genes were related to a wide range of functions and no gene family could be highlighted. Among the up-regulated genes, 166 were transcription factors, the most representative belonging to the Zinc finger RING/FYVE/PHD-type family (22) and WRKY family (19); other genes were from the kinase family, participating in biological processes of protein amino acid phosphorylation (86); had molecular function of iron ion binding (56); were involved in response to oxidative stress (scavenging of reactive oxygen species) (26); had molecular function of transport activity (84), including four genes related to heavy metal transport/detoxification and four genes of the multi antimicrobial extrusion protein MATE family; and were involved in the biological process of apoptosis (14), including 10 genes of NB-ARC. Among the up-regulated genes, 435 present at least one cis-regulatory element responsive to abscisic acid (ABA) with significant occurrence (P≤0.05) in its promoter region (1 kbp upstream of the transcription start site). These data indicate that about 28% of the up-regulated genes can be regulated by changing in the ABA content in leaves in response to iron excess. Regarding expression of LTR retrotransposons, 302 were down-regulated (53 Ty1/Copia, 172 Ty3/Gypsy and 77 unclassified), and 4342 up-regulated (466 Ty1/Copia, 2276 Ty3/Gypsy and 1600 unclassified). They were observed a large activity of LTR retrotransposons in response to iron toxicity, and furthermore, they were verified that LTR retrotransposons transcription can extend to 5' and 3' flanking regions. In addition, 16 situations that should up-regulated LTR retrotransposons are located at a very short distance (smaller than 1000 base pairs) in the same chromosome of up-regulated genes suggesting co-transcription, these occurrences are represented by eight where the LTR retrotransposon and the gene have the same sense of transcription (plus); five occurrences with the both with the same sense of transcription (minus) and one occurrence where they have opposite senses. Additionally, two occurrences that in which both, DNA sequences of up-regulated retrotransposon and gene, are overlapped and have the same sense of transcription. / A toxidez por ferro em plantas está associada com a presença de grandes concentrações de ferro (Fe) reduzido (Fe2+) na solução do solo, esta condição pode ocorrer em solos irrigados por inundação. Os sintomas de toxidez por ferro incluem estresse oxidativo nas folhas como resultado do excesso de Fe2+ absorvido pelas raízes, resultando em perdas na produtividade. As respostas das plantas às condições de estresse envolvem a percepção dos estímulos, transdução de sinais e ativação da transcrição gênica. Além da expressão gênica, os LTR retrotransposons (Long Terminal Repeat Retrotransposons) que respresentam cerca de 20% do genoma do arroz, podem ser transcricionalmente ativados em condições de estresse e desta forma, influenciar a expressão de genes adjacentes (por exemplo devido a alterações na estrutura da cromatina). Este estudo teve por objetivo identificar genes e LTR retrotransposons diferencialmente expressos em plântulas de arroz (Oryza sativa ssp. japonica cv. Nipponbare), após quatro dias de exposição ao excesso de ferro em solução nutritiva. A expressão diferencial de genes e LTR retrotransposons foi analisada utilizando a técnica de microarranjo e sua validação foi realizada por meio de RT-qPCR. O RNA total foi extraído de folhas de plântulas de arroz cv. Nipponbare, após quatro dias de cultivo em solução nutritiva adicionada de ferro na concentração de 7 mM (FeSO47H2O) (presença de toxidez) e a condição controle com presença de ferro na concentração de 10 μM. O cDNA fita dupla foi sintetitizado a partir do RNA mensageiro. A hibridização foi realizada entre o cDNA das duas condições em triplicatas biológicas e o microarranjo Rice Transposome Array v. 2.0 (Roche/NimbleGen technology, an improvement of v.1.0, Picault et al., 2009). Os valores de intensidade de cada spot foram normalizados, transformados e comparados pelo teste T Bayesiano. A identificação dos genes e LTR retrotransposons foi realizada de acordo com o banco de dados RAP-DB (Rice Annotation Project Database, build 5). Considerando log2 FC (log2-fold-change) ≤ -1 como subexpressão e ≥ 1 como superexpressão e P≤ 0.05 para ambas condições. Foram identificados 44 genes subexpressos e 1.572 superexpressos com funções descritas. Os genes subexpressos desempenham a uma vasta gama de funções. Entre elas destacam-se: 166 genes que são fatores de transcrição, sendo que os mais representativos pertencem à família Zinc finger RING/FYVE/PHD-type family (22 genes) e WRKY (19 genes); outros genes da família das cinases que participam também da sinalização celular em processos biológicos de fosforilação de aminoácidos nas proteínas (86 genes); outros genes com função molecular de ligação ao íon ferro (56 genes); 26 genes envolvidos na resposta ao estresse oxidativo (scavengers de espécies reativas de oxigênio); 84 genes com função molecular de transporte, incluindo quatro genes relacionados ao transporte e detoxificação de metais pesados e quatro genes da família MATE; 14 genes envolvidos em apoptose, incluindo 10 genes NB-ARC. Entre os genes superexpressos, 435 apresentam pelo menos um elemento regulatório de ação cis responsivo ao ácido abscisico (ABA) com ocorrência significativa (P≤0,05) em sua região promotora (1 kbp a montante do sítio de início da transcrição). Estes dados indicam que cerca de 28% dos genes superexpressos podem ser regulados pelas alterações no conteúdo de ABA nas folhas, em resposta ao estresse por excesso de ferro. Considerando a expressão do LTR retrotransposons, 302 apresentaram subexpressão (53 Ty1/Copia, 172 Ty3/Gypsy e 77 não classificados), e 4.342 apresentaram superexpressão (466 Ty1/Copia, 2276 Ty3/Gypsy e 1600 não classificados). Foi constatada grande atividade transcricional dos LTR retrotransposons em resposta à toxidez por ferro, sendo que a transcrição dos LTR retrotransposons pode se estender às suas regiões flanqueadoras 5 e 3 , além disso foram encontradas 16 ocorrencias em que o LTR retrotransposon e o gene superexpresso estão localizados a uma distância menor do que 1000 pares de bases no mesmo cromossomo, sugerindo co-transcrição entre ambos. Entre as 16 ocorrências, oito em que o LTR retrotransposon e o gene apresentam o mesmo sentido de transcrição (plus); cinco ocorrências com mesmo sentido de transcrição (minus) e uma ocorrência onde LTR retrotrotransposon e gene apresentam sentidos de transcrição opostos. Foram observadas ainda, duas ocorrências em que as sequencias de DNA do LTR retrotransposon e do gene superexpressos estão sobrepostas, e apresentam o mesmo sentido de transcrição.

Made available in DSpace on 2014-08-20T13:25:37Z (GMT). No. of bitstreams: 1 tese_taciane_finatto.pdf: 5834731 bytes, checksum: e10f781234d54582cc17a9b8dff16c53 (MD5) Previous issue date: 2012-02-27 / Iron toxicity in plants is associated with the presence of large concentrations of reduced iron (Fe2+) in the soil solution, which occurs in flooded soils and affects rice plants grown under this condition. Symptoms of iron toxicity involve oxidative stress in leaves, as a response to excessive Fe2+ absorption by the roots. The responses of plants to stress conditions include stimulus perception, signal transduction and gene transcription activation. Besides gene expression, LTR (Long Terminal Repeat) retrotransposons represent ca. 22% of the rice genome, they can be transcriptionally activated under stress, and they can alter the expression of adjacent genes (e.g. due to alterations in chromatin structure). This study aimed to identify differentially expressed genes and LTR retrotransposons in leaves of 18-day-old rice seedlings (Oryza sativa ssp. japonica cv. Nipponbare) after four days of iron excess exposure. They were identified a differential expression of genes and LTR retrotransposons in rice exposed to iron excess using a microarray approach. Total RNA was extracted from leaves of 18-day-old rice seedlings (Oryza sativa L. ssp japonica cv. Nipponbare) after four days of cultivation in nutrient solution with iron excess (7 mM of FeSO47H2O) and in a control solution. The hybridization was performed with cDNA and rice transposome array v. 2.0 microarray (Roche/NimbleGen technology, an improvement of v.1.0, Picault et al., 2009). Data from gene expression was analyzed by the Bayesian t-test with BH adjustment method. Gene annotation, gene ontology, and LTR retrotransposon identification were performed at RAP-DB (Rice Annotation Project Database, build 5), and microarray results were validated by RT-qPCR. Considering log2 FC (log2-fold-change) ≤ -1 as underexpression and ≥ 1 as overexpression (p-values ≤ 0.05), 44 down-regulated and 1,572 up-regulated genes with described function were identified. Down-regulated genes were related to a wide range of functions and no gene family could be highlighted. Among the up-regulated genes, 166 were transcription factors, the most representative belonging to the Zinc finger RING/FYVE/PHD-type family (22) and WRKY family (19); other genes were from the kinase family, participating in biological processes of protein amino acid phosphorylation (86); had molecular function of iron ion binding (56); were involved in response to oxidative stress (scavenging of reactive oxygen species) (26); had molecular function of transport activity (84), including four genes related to heavy metal transport/detoxification and four genes of the multi antimicrobial extrusion protein MATE family; and were involved in the biological process of apoptosis (14), including 10 genes of NB-ARC. Among the up-regulated genes, 435 present at least one cis-regulatory element responsive to abscisic acid (ABA) with significant occurrence (P≤0.05) in its promoter region (1 kbp upstream of the transcription start site). These data indicate that about 28% of the up-regulated genes can be regulated by changing in the ABA content in leaves in response to iron excess. Regarding expression of LTR retrotransposons, 302 were down-regulated (53 Ty1/Copia, 172 Ty3/Gypsy and 77 unclassified), and 4342 up-regulated (466 Ty1/Copia, 2276 Ty3/Gypsy and 1600 unclassified). They were observed a large activity of LTR retrotransposons in response to iron toxicity, and furthermore, they were verified that LTR retrotransposons transcription can extend to 5' and 3' flanking regions. In addition, 16 situations that should up-regulated LTR retrotransposons are located at a very short distance (smaller than 1000 base pairs) in the same chromosome of up-regulated genes suggesting co-transcription, these occurrences are represented by eight where the LTR retrotransposon and the gene have the same sense of transcription (plus); five occurrences with the both with the same sense of transcription (minus) and one occurrence where they have opposite senses. Additionally, two occurrences that in which both, DNA sequences of up-regulated retrotransposon and gene, are overlapped and have the same sense of transcription. / A toxidez por ferro em plantas está associada com a presença de grandes concentrações de ferro (Fe) reduzido (Fe2+) na solução do solo, esta condição pode ocorrer em solos irrigados por inundação. Os sintomas de toxidez por ferro incluem estresse oxidativo nas folhas como resultado do excesso de Fe2+ absorvido pelas raízes, resultando em perdas na produtividade. As respostas das plantas às condições de estresse envolvem a percepção dos estímulos, transdução de sinais e ativação da transcrição gênica. Além da expressão gênica, os LTR retrotransposons (Long Terminal Repeat Retrotransposons) que respresentam cerca de 20% do genoma do arroz, podem ser transcricionalmente ativados em condições de estresse e desta forma, influenciar a expressão de genes adjacentes (por exemplo devido a alterações na estrutura da cromatina). Este estudo teve por objetivo identificar genes e LTR retrotransposons diferencialmente expressos em plântulas de arroz (Oryza sativa ssp. japonica cv. Nipponbare), após quatro dias de exposição ao excesso de ferro em solução nutritiva. A expressão diferencial de genes e LTR retrotransposons foi analisada utilizando a técnica de microarranjo e sua validação foi realizada por meio de RT-qPCR. O RNA total foi extraído de folhas de plântulas de arroz cv. Nipponbare, após quatro dias de cultivo em solução nutritiva adicionada de ferro na concentração de 7 mM (FeSO47H2O) (presença de toxidez) e a condição controle com presença de ferro na concentração de 10 μM. O cDNA fita dupla foi sintetitizado a partir do RNA mensageiro. A hibridização foi realizada entre o cDNA das duas condições em triplicatas biológicas e o microarranjo Rice Transposome Array v. 2.0 (Roche/NimbleGen technology, an improvement of v.1.0, Picault et al., 2009). Os valores de intensidade de cada spot foram normalizados, transformados e comparados pelo teste T Bayesiano. A identificação dos genes e LTR retrotransposons foi realizada de acordo com o banco de dados RAP-DB (Rice Annotation Project Database, build 5). Considerando log2 FC (log2-fold-change) ≤ -1 como subexpressão e ≥ 1 como superexpressão e P≤ 0.05 para ambas condições. Foram identificados 44 genes subexpressos e 1.572 superexpressos com funções descritas. Os genes subexpressos desempenham a uma vasta gama de funções. Entre elas destacam-se: 166 genes que são fatores de transcrição, sendo que os mais representativos pertencem à família Zinc finger RING/FYVE/PHD-type family (22 genes) e WRKY (19 genes); outros genes da família das cinases que participam também da sinalização celular em processos biológicos de fosforilação de aminoácidos nas proteínas (86 genes); outros genes com função molecular de ligação ao íon ferro (56 genes); 26 genes envolvidos na resposta ao estresse oxidativo (scavengers de espécies reativas de oxigênio); 84 genes com função molecular de transporte, incluindo quatro genes relacionados ao transporte e detoxificação de metais pesados e quatro genes da família MATE; 14 genes envolvidos em apoptose, incluindo 10 genes NB-ARC. Entre os genes superexpressos, 435 apresentam pelo menos um elemento regulatório de ação cis responsivo ao ácido abscisico (ABA) com ocorrência significativa (P≤0,05) em sua região promotora (1 kbp a montante do sítio de início da transcrição). Estes dados indicam que cerca de 28% dos genes superexpressos podem ser regulados pelas alterações no conteúdo de ABA nas folhas, em resposta ao estresse por excesso de ferro. Considerando a expressão do LTR retrotransposons, 302 apresentaram subexpressão (53 Ty1/Copia, 172 Ty3/Gypsy e 77 não classificados), e 4.342 apresentaram superexpressão (466 Ty1/Copia, 2276 Ty3/Gypsy e 1600 não classificados). Foi constatada grande atividade transcricional dos LTR retrotransposons em resposta à toxidez por ferro, sendo que a transcrição dos LTR retrotransposons pode se estender às suas regiões flanqueadoras 5 e 3 , além disso foram encontradas 16 ocorrencias em que o LTR retrotransposon e o gene superexpresso estão localizados a uma distância menor do que 1000 pares de bases no mesmo cromossomo, sugerindo co-transcrição entre ambos. Entre as 16 ocorrências, oito em que o LTR retrotransposon e o gene apresentam o mesmo sentido de transcrição (plus); cinco ocorrências com mesmo sentido de transcrição (minus) e uma ocorrência onde LTR retrotrotransposon e gene apresentam sentidos de transcrição opostos. Foram observadas ainda, duas ocorrências em que as sequencias de DNA do LTR retrotransposon e do gene superexpressos estão sobrepostas, e apresentam o mesmo sentido de transcrição.estresse oxidativo (scavengers de espécies reativas de oxigênio); 84 genes com função molecular de transporte, incluindo quatro genes relacionados ao transporte e detoxificação de metais pesados e quatro genes da família MATE; 14 genes envolvidos em apoptose, incluindo 10 genes NB-ARC. Entre os genes superexpressos, 435 apresentam pelo menos um elemento regulatório de ação cis responsivo ao ácido abscisico (ABA) com ocorrência significativa (P≤0,05) em sua região promotora (1 kbp a montante do sítio de início da transcrição). Estes dados indicam que cerca de 28% dos genes superexpressos podem ser regulados pelas alterações no conteúdo de ABA nas folhas, em resposta ao estresse por excesso de ferro. Considerando a expressão do LTR retrotransposons, 302 apresentaram subexpressão (53 Ty1/Copia, 172 Ty3/Gypsy e 77 não classificados), e 4.342 apresentaram superexpressão (466 Ty1/Copia, 2276 Ty3/Gypsy e 1600 não classificados). Foi constatada grande atividade transcricional dos LTR retrotransposons em resposta à toxidez por ferro, sendo que a transcrição dos LTR retrotransposons pode se estender às suas regiões flanqueadoras 5 e 3 , além disso foram encontradas 16 ocorrencias em que o LTR retrotransposon e o gene superexpresso estão localizados a uma distância menor do que 1000 pares de bases no mesmo cromossomo, sugerindo co-transcrição entre ambos. Entre as 16 ocorrências, oito em que o LTR retrotransposon e o gene apresentam o mesmo sentido de transcrição (plus); cinco ocorrências com mesmo sentido de transcrição (minus) e uma ocorrência onde LTR retrotrotransposon e gene apresentam sentidos de transcrição opostos. Foram observadas ainda, duas ocorrências em que as sequencias de DNA do LTR retrotransposon e do gene superexpressos estão sobrepostas, e apresentam o mesmo sentido de transcrição.