Дисертації з теми "MiR486"

Genome wide association studies (GWAS) identified the ANK1 gene as a common type 2 diabetes mellitus (T2D) susceptibility locus. More recently, GWAS studies identified a novel SNP associated to T2D susceptibility, namely rs508419, in the internal promoter of ANK1 gene, which drives the expression of small ankyrin 1.5 (sAnk1.5), a striated muscle-specific protein localized in the sarcoplasmic reticulum (SR) membrane. sAnk1.5 interacts with the giant sarcomeric protein obscurin, allowing the correct localization of SR around the myofibrils. The rs508419 SNP is characterized by the substitution of a thymine with a cytosine, which determines an increased transcriptional activity of the ANK1 internal promoter, resulting in high protein levels of sAnk1.5 in skeletal muscle biopsies of individuals carrying the C/C variant of the SNP, with respect to individuals carrying either T/T or C/T genotype. Interestingly, the sequence of microRNA-486 (miR-486), a small non-coding RNA, is positioned in the intron between exon 41 and exon 42 of the coding sequence of sAnk1.5. Therefore, miR-486 is expressed under the transcriptional control of both the principal and of the internal promoters of ANK1. miR-486 plays a role in the regulation of the PI3K/AKT signalling pathway, which regulates several cellular processes, such as growth, cellular proliferation and survival, protein synthesis and degradation, lipid and glucose metabolism. In our laboratory we analysed human skeletal muscle biopsies to evaluate whether miR-486 expression levels were increased in individuals carrying the SNP rs508419. These results showed a significant 3-fold increase of miR-486 expression levels in the skeletal muscle of individuals carrying the C/C variant of the SNP compared to those carrying the T/T variant of the SNP. Given that skeletal muscle is one of the main tissues involved in regulating glucose disposal, the aim of this thesis was to verify whether sAnk1.5 and miR-486 overexpression in mouse skeletal muscle might associate with T2D susceptibility. To this goal, we generated a double transgenic mouse model, (TgsAnk1.5/+//TgmiR486/+), hereafter referred to as double Tg (D-Tg), where the sAnk1.5 coding sequence and the miR-486 sequence are under the transcriptional control of the skeletal muscle-specific rat myosin light chain (MLC) promoter, and of the mouse muscle-specific creatine kinase (CKM) promoter, respectively. Accordingly, the D-Tg mouse was expected to present a skeletal muscle-specific increase of sAnk1.5 and miR-486 expression. Indeed, RT-PCR experiments indicated that the expression of the transgene was restricted to striated muscles and sAnk1.5 and miR-486 mRNAs levels were both robustly increased in transgenic mice compared to WT. Accordingly, western blot analysis of protein extracts from gastrocnemius, 4 extensor digitorum longus (EDL) and soleus muscles revealed an increase in sAnk1.5 protein levels of 45%, 60% and 35%, respectively, with respect to control mice. Notably, in the D-Tg mouse model we observed a significant discrepancy between the levels of sAnk1.5 protein expression and the levels of the corresponding mRNA. Indeed, in the gastrocnemius muscle, sAnk1.5 mRNA was about 12 times more expressed in transgenic mice, compared to only 1.5-fold increase in protein expression, suggesting a post-translational regulation of sAnk1.5 expression. Evidence of this was confirmed by muscular administration of the proteasome inhibitor MG132 in transgenic mice. Results showed sAnk1.5 protein expression levels significantly increased of about 35% compared to those observed in the contralateral untreated gastrocnemius muscles. To evaluate the potential association between sAnk1.5 and miR-486 overexpression with T2D, glucose and insulin tolerance were monitored during a period of 12 months in male transgenic mice. Blood glucose levels after overnight fasting were significantly higher in 2 and 6 months old transgenic mice compared to WT controls. However, overall glucose and insulin tolerance was not altered between the two experimental groups. Finally, considering the miR-486 role in regulating PI3K/AKT signalling pathway, which in skeletal muscle fiber is activated following insulin stimulation thus being a key pathway in maintaining glucose homeostasis, we characterized the expression pattern of PTEN, p85α, FOXO1a, pAKTser473, and GLUT4 in the skeletal muscles of D-Tg mice. Currently, we are performing additional experiments where D-Tg mice are fed with a high-fat diet to better evaluate whether sAnk1.5 and miR-486 overexpression might predispose to T2D susceptibility.

Biology
Ph.D.
Chronic Hepatitis B Virus (HBV) infection is a global health problem because of its connection to acute and chronic liver diseases as well as hepatocellular carcinoma (HCC). There is increasing evidence showing that HBV contributes to HCC due to persistently high levels of trans-activating protein---hepatitis B encoded x antigen (HBxAg). Studies have shown that the HBxAg affects and alters the activity of many different transcription factors and plays an essential role in several cytoplasmic signaling transduction pathways, such as Wnt signaling pathways. One of the upregulated genes, designated URG11, was found transactivated by HBxAg. URG11 could stimulate the ß-catenin promoter and hepatocellular growth and survival which suggest that URG11 may be a regulatory element in the ß-catenin signaling pathways. microRNA148a (miR148a) was identified from two miRNA microarrays as one of the up-regulated miRNAs in cells stably expressing HBxAg or over-expressing URG11. Moreover, the expression of miR148a was also elevated in HBV-mediated HCC patient tissue samples. To study the function of miR148a, HepG2 (hepatoblastoma) and Hep3B (hepatoma) cells stably expressing HBxAg or over-expressing URG11 were transduced by recombinant lentiviruses encoding anti-miR148a. anti-miR148a suppressed cell proliferation, cell cycle progression, cell migration, anchorage independent growth in soft agar and subcutaneous tumor formation in SCID mice. Further, introduction of anti-miR148a increased PTEN protein and mRNA expression, suggesting that PTEN was suppressed by miR148a. In addition, anti-miR148a blocked the stimulation of Akt signaling, resulting in decreased expression of ß-catenin. Thus, miR148a may play a central role in HBxAg/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type.
Temple University--Theses

Orientador: Fábio Silveira Nogueira
Resumo: O sistema radicular (SR) é importante pela ancoragem e obtenção de água e nutrientes. Em eudicotiledôneas, como Arabidopsis, o crescimento da raiz primária (RP) é afetado por fitormônios, especialmente pelo balanço entre auxina que controla a divisão celular, e citocinina que modula a diferenciação celular; também, os microRNAs (miRNAs), um sub-conjunto de pequenos RNAs que regulam pós-transcricionalmente seus alvos, regulam o crescimento da RP. O microRNA156 (miR156) e seus alvos, membros da família SQUAMOSA Promoter-Binding Protein-Like (SPL), constituem uma via genética que regula vários processos do desenvolvimento, incluindo desenvolvimento da raíz; porém, durante o crescimento da RP, não foi observado o efeito da via miR156/SPL, e da interação com auxina e citocinina; assim, foi avaliada essa interação durante o crescimento da PR regulado pelo tamanho do meristema da raiz (TMR) em Arabidopsis. Usando ferramentas genéticas e moleculares foi analizada a expressão de genes MIR156 e SPLs, o comprimento da RP, o TMR, as taxas de divisão celular, e as respostas de auxina e citocinina durante o crescimento da RP. Os genes MIR156 e SPLs possuem padrões de expressão opostos. Níveis altos do miR156 (nas plântulas p35S :: MIR156A), leva a menor comprimento da RP, TMR reduzido, menores taxas de divisão celular, respostas mais baixas e altas à auxina e citocinina respectivamente; em contraste, níveis severamente reduzidos do miR156 maduro disponível (nas plantas MIM156) conducem a e... (Resumo completo, clicar acesso eletrônico abaixo)
Doutor

A genética molecular permite o entendimento dos mecanismos que regulam o desenvolvimento dos órgãos vegetais em resposta a fatores bióticos e abióticos. A regulação de vias gênicas é de fundamental importância no sucesso reprodutivo, evolutivo e econômico dos vegetais. Uma das modalidades de regulação é a pós transcricional através de microRNAs (miRNAs). Tratam-se de pequenos RNAs endógenos não codantes que possuem uma complementaridade quase perfeita em plantas. Em plantas, muitos genes-alvos de miRNA codificam-se para fatores de transcrição, como os genes SQUAMOSA Promoter-Binding Protein-Like (SPL/SBP). O microRNA156, conservado entre as Angiospermas, regula diversos fatores de transcrição da família SBP. O módulo miR156/SBP, denominado via da idade (AGE), atua ao longo do ciclo de vida dos vegetais regulando as transições de fase: juvenil-adulta e vegetativa-reprodutiva. O tomateiro, Solanum lycopersicum L., possui genes SlSBPs regulados pelo miR156 e com funcionalidade não esclarecida. Dentre aqueles, o gene SlSBP6c (Solyc12g038520) se destaca por possuir maior similaridade filogenética com SPL6s/SBP6s de solanáceas do que seus genes homólogos SlSBP6a e SlSBP6b o que sugere a hipótese de um possível ganho de função. Com o intuito de testar essa hipótese, o presente trabalho objetivou caracterizar funcionalmente o gene SlSBP6c. Para tanto, utilizou-se o genótipo 35S::rSlSBP6c, planta transformada de tomateiro cultivar Micro-Tom (MT) que super-expressa a versão resistente ao miR156 do gene SlSBP6, fusionada ao promotor viral 35S. Este material vegetal foi gerado pelo laboratório de Genética Molecular do Desenvolvimento Vegetal e cedido para execução desta pesquisa. O trabalho se desenvolveu em duas etapas: caracterização molecular e morfo-fisiológica. Primeiramente, os genes SlSBP6a, SlSBP6b e SlSBP6c de tomateiro foram analizados quanto a sua expressão ao longo do desenvolvimento em folhas e inflorescência. E, posteriormente, foram analisados a complexidade foliar, o tempo de florescimento, a transição do meristema vegetativo para o reprodutivo, o teor relativo de clorofila e a fotossíntese líquida. Os resultados obtidos demonstram que os genes SlSBP6a e SlSBP6c são semelhantes no padrão de expressão gênica na homologia das protéinas que o codificam, indicando similaridade funcional. A de-regulação do gene SlSBP6c leva ao aumento a complexidade foliar, o teor relativo de clorofila e a fotossíntese líquida. O atraso na transição do meristema vegetativo para o reprodutivo se evidencia por um florescimento tardio nas plantas que super-expressam o gene SlSBP6c. Os resultados demonstram que o gene SlSBP6c exerce funcionalidade no tomateiro atuando na transição de fase vegetativo-reprodutivo.
Molecular genetics allows the understanding of the mechanisms that regulate the development of plant organs in response to biotic and abiotic factors. The regulation of gene pathways is of fundamental importance in the reproductive, evolutionary and economic success of the plants. Research has been increasing the knowledge about post-transcriptional regulation through microRNAs (miRNAs). The miRNAs are small non-coding endogenous RNAs that have almost perfect complementarity in plants. Many miRNA target genes in plants are encoded by transcription factors, such as the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL / SBP) genes. MicroRNA156 is conserved among Angiosperms and regulates several transcription factors of the SBP family. The miR156 / SBP module, called the age pathway (AGE), acts throughout the life cycle of plants regulating phase transitions juvenile-adult and vegetative-reproductive. The tomato, Solanum lycopersicum L., has newly described and unintelligently regulated miR156 regulated SlSBPs. Among these, the gene SlSBP6c (Solyc12g038520) stands out for having greater phylogenetic similarity with solanaceous SBP6s than its homologous genes SlSBP6a and SlSBP6b, indicating a possible gain of function related to characteristics of this family of plants as fleshy fruits and composite leaves. In order to test this hypothesis the work aimed to characterize the SlSBP6c gene functionally. Using as a tool the 35S::rSlSBP6c genotype, transformed tomato plant micro-Tom (MT) that over-expresses the miR156 resistant version of the SlSBP6 gene, fused to the 35S viral promoter. The Laboratory of Molecular Genetics of Plant Development generated this plant material. The work was developed in two stages: molecular and morpho-physiological characterization. In the first the genes SlSBP6a, SlSBP6b and SlSBP6c of tomato were analyzed for their expression throughout the development in leaves and inflorescence. In the second, the leaf complexity, the flowering time, the transition from vegetative to the reproductive meristem, the relative chlorophyll content and the net photosynthesis were evaluated. The results obtained demonstrate that the SlSBP6a and SlSBP6c genes are very similar in the pattern of gene expression in the homology of the coding proteins, indicating a functional similarity. The SlSBP6c gene acts to increase leaf complexity, relative chlorophyll content and liquid photosynthesis. A late flowering in plants that overexpress the SlSBP6c gene evidences the delay in the transition from the vegetative to the reproductive meristem. The data set demonstrates that the SlSBP6c gene has important functionality in tomatoes acting on vegetative-reproductive phase transition.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A cana-de-açúcar é atualmente uma das principais culturas da agroindústria mundial. Devido à ampla expansão de seu plantio é submetida constantemente a solos não produtivos. A presença de moléculas tóxicas no solo, como o alumínio (Al3+), interfere diretamente no desenvolvimento radicular ocasionando baixa absorção de água e nutrientes levando a pouca produtividade e desenvolvimento das plantas. Os microRNAs tem sido descritos como um dos fatores responsáveis pela regulação gênica e a descoberta dessas moléculas abre um novo caminho para a elucidação da tolerância e adaptação das plantas aos estresses abióticos. Este estudo visou avaliar em duas cultivares de cana-de-açúcar (CTC-2 e RB855453) a expressão dos microRNAs miR159, miR164 e miR168, associados à resposta ao alumínio em espécies como Arabidopsis thaliana, arroz (Oriza sativa) e tabaco (Nicotiana tabacum). Algumas características foram avaliadas, como densidade de raízes (DS), área foliar (AR), produção de massa seca (MS) e teor de prolina nas folhas, em quatro cultivares de cana-de-açúcar submetidas a diferentes concentrações de alumínio. A cultivar CTC 2 foi classificada como tolerante e a RB855453 como sensível ao estresse. Ambas foram selecionadas e em sistema de hidroponia submetidas novamente ao estresse pela toxidez de alumínio na concentração de 221 μmol L -1 . Parâmetros fisiológicos foram mensurados (área foliar, potencial osmótico, taxa de fotossíntese, transpiração, condutância estomática) e a expressão dos miRNAs 159, 164 e 168 avaliada por PCR em tempo real. Plantas das duas cultivares apresentaram alterações fisiológicas e morfológicas ao longo do estresse, com redução significativa para a área foliar da cultivar RB855453. O miR164 e 159 foram induzidos nas duas cultivares, principalmente após 72 horas de estresse, e o miR168 diferencialmente expresso. Esses miRNAs regulam genes e fatores de transcrição que estão envolvidos na resposta e desenvolvimento da planta diante ao estresse por alumínio.
The sugarcane is currently one of the main crops of global agribusiness. Due to the wide expansion of its plantation is constantly subjected unproductive soils. The presence of toxic molecules in the soil, such as aluminum (Al3+), directly affects root development, leading to poor absorption of nutrients and water leading to low productivity and development of plants. Studies of the interactions of plants with the environment are being conducted to clarify the resistance or susceptibility of various cultures, favoring the discovery of important mechanisms that participate in physiological and molecular responses to environmental stresses. MicroRNAs have been described as one of the factors responsible for gene regulation and the discovery of these molecules opens a new path for the elucidation of tolerance and adaptation of plants to abiotic stresses. This study evaluated in two sugarcane varieties the expression of microRNA miR159, miR164 and miR168, associated with the response to the aluminum species such as Arabidopsis thaliana, rice (Oryza sativa) and tobacco (Nicotiana tabacum). Some characteristics were evaluated, such as density (DS), area (AR), dry mass production (DM) and proline content, in four sugarcane cultivars submitted to different concentrations of aluminum. CTC 2 cultivar was classified as tolerant and RB855453 as stress sensitive. Both were selected and in a hydroponics system again submitted to stress by the aluminum toxicity in the concentration of 221 μmol L-1. Physiological parameters were measured (leaf area, osmotic potential, photosynthesis rate, transpiration, stomatal conductance) and the expression of miRNAs 159, 164 and 168 evaluated by real-time PCR. Plants of both cultivars presented physiological and morphological changes along the stress, with a significant reduction for the leaf area of cultivar RB855453. The miR164 and 159 were induced in the two cultivars, mainly after 72 hours of stress, and the miR168 differentially expressed. These miRNAs regulate genes and transcription factors that are involved in the response and development of the plant in the face of aluminum stress.

Os microRNAs (miRNAs) são pequenos RNAs endógenos não codantes de 21-24 nucleotídeos (nt) que regulam a expressão gênica de genes-alvos. Eles estão envolvidos em diversos aspectos de desenvolvimento da planta, tanto na parte aérea, quanto no sistema radicular. Entre os miRNAs, o miRNA156 (miR156) regula a família de fatores de transcrição SQUAMOSA Promoter-Binding Protein-Like (SPL) afetando diferentes processos do desenvolvimento vegetal. Estudos recentes mostram que a via gênica miR156/SPL apresenta efeito positivo tanto no aumento da formação de raízes laterais, quanto no aumento de regeneração de brotos in vitro a partir de folhas e hipocótilos em Arabidopsis thaliana. Devido ao fato de que a origem da formação de raiz lateral e a regeneração in vitro de brotos a partir de raiz principal compartilham semelhanças anatômicas e moleculares, avaliou-se no presente estudo se a via miR156/SPL, da mesma forma que a partir de explantes aéreos, também é capaz de influenciar na regeneração de brotos in vitro a partir de explantes radiculares. Para tanto foram comparados taxa de regeneração, padrão de distribuição de auxina e citocinina, análises histológicas e histoquímicas das estruturas regeneradas em plantas com via miR156/SPL alterada, incluindo planta mutante hyl1, na qual a produção desse miRNA é severamente reduzida. Além disso, foi avaliado o padrão de expressão do miR156 e específicos genes SPL durante a regeneração de brotos in vitro a partir da raiz principal de Arabidopsis thaliana. No presente trabalho observou-se que a alteração da via gênica miR156/SPL é capaz de modular a capacidade de regeneração de brotos in vitro a partir de raiz principal de Arabidopsis thaliana e a distribuição de auxina e citocinina presente nas células e tecidos envolvidos no processo de regeneração. Plantas superexpressando o miR156 apresentaram redução no número de brotos regenerados, além de ter o plastochron reduzido quando comparado com plantas controle. Adicionalmente, plantas contento o gene SPL9 resistente à clivagem pelo miR156 (rSPL9) apresentaram severa redução na quantidade de brotos, além de terem o plastochron alongado. Interessantemente, plantas mutantes hyl1-2 e plantas rSPL10 não apresentaram regeneração de brotos ao longo da raiz principal, mas sim intensa formação de raízes laterais e protuberâncias, respectivamente, tendo essa última apresentado indícios de diferenciação celular precoce. Tomados em conjunto os dados sugerem que o miR156 apresenta importante papel no controle do processo de regeneração de brotos in vitro. Entretanto, esse efeito é mais complexo em regeneração in vitro a partir de raízes do que a partir de cotilédones ou hipocótilos.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs of 21-24 nucleotides (nt) in length that regulate target gene expression. They are involved in many aspects of plant development, both in the shoot and in the root systems. Among miRNAs, miRNA156 (miR156) regulates SQUAMOSA Promoter Binding-Like (SPL) transcription factor family affecting different plant development processes. Recent studies have shown that the miR156/SPL pathway has a positive effect both in the increase of lateral root formation and regeneration of shoots from leaves and hypocotyls in Arabidopsis thaliana. Because the origin of lateral root formation and in vitro shoot regeneration from primary root share similar anatomical and molecular features, in the present study was evaluated whether the miR156/SPL pathway, in the same manner that from aerial explants, is also able to influence the in vitro shoot regeneration from root explants. For this, it was compared regeneration rates, distribution pattern of auxin and cytokinin, histological and histochemical analyses of the structures regenerated in plants in with the miR156/SPL pathway is modified, including the mutant hyl1-1, in which the biosynthesis of this miRNA is severely reduced. Besides that, it was evaluated the expression pattern of miR156 and specific SPL target genes during in vitro shoot regeneration from primary roots of Arabidopsis it was observed that the alteration on the miR156/SPL pathway is capable to modulate in vitro shoot regeneration from the primary root of Arabidopsis and the distribution of auxin and cytokinin at the tissues and cells involved in the regeneration process. Plants overexpressing the miR156a have shown reduction in the number of regenerated shoots, and displayed a reduction in plastochron when compared with wild type plants. Additionally, plants expressing cleavage-resistant form of SPL9 (rSPL9) presented severe reduction in the amount of shoots, and extended plastochron. Interestingly, mutant hyl1-2 and plants rSPL10 did not show any shoot regeneration along the root, but high formation of lateral roots and protuberances, respectively, having rSPL10 presented evidence of precocious cell differentiation. Taken together, these data suggest that de miR156 and SPLs have an important role in the control the in vitro shoot regeneration process. However, its effect is somehow more complex in roots than in cotyledons or hypocotyls.

Les petits ARN non codant et particulièrement les microARN ont été récemment impliqués comme régulateurs post-transcriptionnels du développement et de la réponse aux stress chez les plantes. Les travaux présentés ici avaient pour but d’identifier et de caractériser de nouveaux petits ARN qui pourraient intervenir dans la réponse aux contraintes environnementales et/ou la plasticité de l’architecture racinaire. L’analyse d’une banque de petits ARN, construite à partir de cultures cellulaires d’Arabidopsis thaliana traitées au H2O2, a permis d’identifier 51 nouvelles séquences dont certaines présentent des caractéristiques particulières et/ou des domaines d’expression différents dans la plante ou qui pourraient être liés au stress oxydatif. Des cibles potentielles de ces petits ARN ont été identifiées. De plus, nous avons identifié et caractérisé un microARN spécifique d’Arabidopsis, le MIR773, s’accumulant préférentiellement dans les racines. Il cible certaines protéines de la famille MET, qui sont les homologues végétaux de la cytosine ADN méthyltransférase majeure chez les animaux (Dnmt1). Nous avons généré des lignées transgéniques qui surexpriment ce MIR et analysé en détail leur développement ainsi que leurs profils de méthylation. Toutefois, nos données indiquent que ce couple microARN/cible n’interviendrait pas dans la régulation de la méthylation de l’ADN. Enfin, nous décrivons le rôle du gène MtMIR166a, qui contient deux copies en tandem du MIR166, dans la régulation de plusieurs facteurs de transcription de type class III HD-ZIP (HomeoDomain leucine-ZIPper) intervenant dans le développement des nodules et des racines secondaires chez Medicago truncatula
Small non coding RNAs, and particularly microRNAs, have been lately implicated as post-transcriptional regulators of several developmental processes and stress responses in plants. This project aimed to identify and characterize new small RNAs that could be involved in responses to environmental constraints and/or linked to the root adaptative plasticity. Using a small RNA library constructed from Arabidopsis thaliana cell cultures treated with H2O2, we identified 51 new small RNA sequences. Among them, several displayed particular characteristics and/or expression patterns in plant tissues or in response to oxidative stress. Potential targets were identified. Furthermore, we identified and characterized one Arabidopsis-specific microRNA, MIR773, whose expression is enriched in root tissues. MIR773 targets a subset of the MET family proteins, which are plant homologues of the major DNA cytosine methyltransferase in mammals (Dnmt1) and plants (MET1). We generated transgenic lines overexpressing this microRNA and analyzed in detail their phenotypes during development and in response to abiotic stresses. Finally, we attempted to analyze the methylation profiles of these lines. However, results indicate that this microRNA/target pair may not be involved in the regulation of DNA methylation in plants. Finally, we describe the involvement of the MtMIR166a locus, which contains two tandem copies of mature MIR166, in the regulation of several class III HD-ZIP (HomeoDomain leucine-ZIPper) genes in Medicago truncatula. Particularly, this microRNA has been involved in the regulation of symbiotic nodule and lateral root development

O florescimento é um processo chave no desenvolvimento vegetal. A mudança de identidade do meristema apical de vegetativo para reprodutivo desencadeia reprogramação genética com efeitos em todo o corpo vegetal. Arabidopsis thaliana é conhecida como o principal modelo de estudo para esse processo apresentando até o momento cinco principais vias genéticas regulatórias. Tais vias apresentam redundância, sendo complexa a eliminação total da transição de fase nessa espécie. A via AGE, regulada pela idade da planta, tem como principais reguladores o mir156 e seus alvos diretos, os fatores de transcrição da família SPL/SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like). Uma segunda via é controlada pelo fitohormônio giberelina (GA), o qual atua de maneira oposta em Arabidopsis thaliana (arabidopsis) e Solanum lycopersicum L. (tomateiro). Em tomateiro, diferentemente de arabidopsis, o cruzamento entre mutantes com conteúdo alterado de GA e plantas transgênicas superexpressando o miR156 (156OE; SILVA et al., 2014) demonstraram efeito sinérgico no atraso do tempo de florescimento. A aplicação de GA3 em plantas 156OE apresenta efeito similar aos cruzamentos citados sobre a transição do meristema apical. Em um dos cruzamentos entre mutantes da via GA e plantas 156OE, foi possível obter plantas apresentando completo bloqueio da transição de fase vegetativo-reprodutivo. A oferta extra do florígeno SINGLE FLOWER TRUSS (SFT) via enxertia não foi suficiente para restaurar a transição de fase nessas plantas, sugerindo que vias associadas à GA e AGE regulam alvos em comum, os quais podem ser independentes da regulação por SFT. Além disso, a regulação transcricional, e possivelmente pós-transcricional de alguns genes SBPs por diferentes vias associadas à GA, sugere uma complexa inter-relação entre as vias GA e AGE em tomateiro durante o florescimento. A ação combinada das vias GA e AGE foi capaz de inibir completamente o florescimento em tomateiro, regulação oposta ao verificado na planta modelo Arabidopsis thaliana. O efeito inibitório de GA sobre o florescimento é também visualizado em plantas lenhosas, sugerindo que as descobertas científicas realizadas em tomateiro podem ser expandidas para essas espécies, nas quais a experimentação é lenta e laboriosa
The flowering process is a major developmental event during the plant life cicle. The meristem identity switches from vegetative to reproductive, triggering substantial genetic modifications that affect the whole plant body. Arabidopsis thaliana is a major model for flowering with five different pathways controlling this process. These pathways are redundant, making complex the complete elimination of phase change in this species. One of the pathways is termed AGE since it is regulated by the time of development. The miR156 and its direct target SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like) are the main regulators of the AGE pathway. A second pathway is controlled by the phytohormone gibberellin (GA), which acts in opposite ways when comparing Arabidopsis thaliana and tomato. In tomato, unlike Arabidopsis, the cross between mutants with altered contents of GA and transgenic plants overexpressing the miR156 (156OE; SILVA et al, 2014) showed synergistic effect in delayed flowering time. Treatments of GA3 in plants 156OE lead to similar effects visualized on the crosses above related to meristem transition. Among the crosses between GA mutants and 156OE plants, one double mutant could completely abolish the phase change in tomato. An extra offer of the florigen (SINGLE FLOWER TRUSS or SFT) by grafting experiments was unable to restore the flowering process in this double mutant. It suggests, pathways associated to GA and AGE regulate common downstream targets, which could be independent of SFT regulation. Moreover, the transcriptional regulation, and possible the post-transcriptionally regulation of some SBP targets by different pathways associated to GA, suggest a complex network between GA and AGE during the flowering in tomato. The combined action of GA and AGE pathways can complete impaired the flowering in tomato, this interaction is opposed to the model Arabidopsis thaliana. The negative effect of GA over the time of flowering is presented in wood plants, suggesting the scientific discoveries in tomato could be expanded to these species, which experiments are slow and laborious

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O desenvolvimento das plantas depende da atividade de um grupo de células em divisão chamado de meristema. Extensas análises genéticas identificaram os principais reguladores do meristema apical vegetativo (SAM), os quais controlam o desenvolvimento de todos os órgãos aéreos. Dentre eles, há um grupo de homeoproteínas denominadas TALE (three-amino-acid-loop- extension); esta família contém os membros KNOTTED-like homeodomain (KNOX) e BELL-like Homeodomain (BELL), que funcionam como homodímeros ou heterodímeros, para regular a expressão de seus genes alvos mediante sua ligação à sequências especificas no DNA. Em plantas com folhas compostas como o tomateiro (Solanum lycopersicum), genes KNOX da classe I (KNOX I) são expressos no meristema, assim como também em folhas, flores e frutos, sugerindo que eles podem exercer várias funções nestes órgãos. Esta hipótese é corroborada pelos fenótipos intrigantes encontrados em mutantes com ganho de função dos genes KNOX I, cuja expressão ectópica afeta a forma da folha, pétala e frutos. Um exemplo é o tomateiro mutante Mouse ear (Me), que superexpressa o gene TKN2 (KNOX I). Fenótipos semelhantes também foram observados em plantas transgênicas superexpressando o microRNA156 (miR156). Os MicroRNAs são uma nova classe de pequenas moléculas de RNA não codantes (20-25 nucleotídeos) que se encontram amplamente distribuídos no genoma de plantas e animais, regulando a expressão de seus genes alvos principalmente ao nível pós-transcricional. O miR156 regula pós-transcricionalmente membros da família gênica do tipo SQUAMOSA Promoter-Binding Protein-Like (SPL ou SBP-box), os quais codificam fatores de transcrição específicos de plantas. Tais genes desempenham papéis importantes em diferentes aspectos do desenvolvimento. Para analisar a possível interação molecular entre o fator de transcrição TKN2 e a via microRNA156/SQUAMOS Promoter-Binding ...
Plant development depends on the activity of a group of dividing cells called meristem. Extensive genetic analyses have identified the major regulators of the shoot apical meristem (SAM), which control the development of all aerial organs. Among them, the three-amino-acid- loop-extension (TALE) class of homeoproteins; this family contains the KNOTTED-like homeodomain (KNOX) and BELL-like Homeodomain (BELL) members, which function as heterodimers or homodimers, to regulate expression of their target genes by binding to specific sequences in DNA. In plants with compound leaves as tomato (Solanum lycopersicum), KNOX I are expressed in the meristem, as well as on leaves, flowers and fruits, suggesting that they may play various roles in these organs. This hypothesis is supported by the intriguing phenotypes found in mutants with gain-of function of KNOX I genes, whose ectopic expression affects leaf, petal and fruit shape. An example, is the tomato mutant Mouse ear (Me), which overexpress the gene TKN2 (KNOX I). Similar phenotypes were also observed in transgenic plants overexpressing microRNA156 (miR156). MicroRNAs are a class of small no-coding RNAs (20-25 nucleotides) that are widely distributed in the genome of plants and animals, regulating the expression of their target genes by acting mainly at the post-transcriptional level. miR156 regulated post-transcriptionally most SQUAMOSA Promoter-Binding Protein-Like (SPL or SBP-box) genes, which encode plant-specific transcription factors. These genes play important roles in different aspects of development. To examine a possible molecular interaction between TKN2 transcription factor and microRNA156/SQUAMOSA Promoter-Binding Protein-Like module (miR156 node), it was evaluated the expression of miR156, its targets (SBP-box) and several genes downstream of miR156 node in different stages of the development of homozygous Me plants. Moreover, to evaluate the genetic interaction ...

Orientador: Fábio Tebaldi Silveira Nogueira
Banca: Luiz Fernando Rolim de Almeida
Banca: Edson Luiz Furtado
Resumo: O desenvolvimento das plantas depende da atividade de um grupo de células em divisão chamado de meristema. Extensas análises genéticas identificaram os principais reguladores do meristema apical vegetativo (SAM), os quais controlam o desenvolvimento de todos os órgãos aéreos. Dentre eles, há um grupo de homeoproteínas denominadas TALE (three-amino-acid-loop- extension); esta família contém os membros KNOTTED-like homeodomain (KNOX) e BELL-like Homeodomain (BELL), que funcionam como homodímeros ou heterodímeros, para regular a expressão de seus genes alvos mediante sua ligação à sequências especificas no DNA. Em plantas com folhas compostas como o tomateiro (Solanum lycopersicum), genes KNOX da classe I (KNOX I) são expressos no meristema, assim como também em folhas, flores e frutos, sugerindo que eles podem exercer várias funções nestes órgãos. Esta hipótese é corroborada pelos fenótipos intrigantes encontrados em mutantes com ganho de função dos genes KNOX I, cuja expressão ectópica afeta a forma da folha, pétala e frutos. Um exemplo é o tomateiro mutante Mouse ear (Me), que superexpressa o gene TKN2 (KNOX I). Fenótipos semelhantes também foram observados em plantas transgênicas superexpressando o microRNA156 (miR156). Os MicroRNAs são uma nova classe de pequenas moléculas de RNA não codantes (20-25 nucleotídeos) que se encontram amplamente distribuídos no genoma de plantas e animais, regulando a expressão de seus genes alvos principalmente ao nível pós-transcricional. O miR156 regula pós-transcricionalmente membros da família gênica do tipo SQUAMOSA Promoter-Binding Protein-Like (SPL ou SBP-box), os quais codificam fatores de transcrição específicos de plantas. Tais genes desempenham papéis importantes em diferentes aspectos do desenvolvimento. Para analisar a possível interação molecular entre o fator de transcrição TKN2 e a via microRNA156/SQUAMOS Promoter-Binding ...
Abstract: Plant development depends on the activity of a group of dividing cells called meristem. Extensive genetic analyses have identified the major regulators of the shoot apical meristem (SAM), which control the development of all aerial organs. Among them, the three-amino-acid- loop-extension (TALE) class of homeoproteins; this family contains the KNOTTED-like homeodomain (KNOX) and BELL-like Homeodomain (BELL) members, which function as heterodimers or homodimers, to regulate expression of their target genes by binding to specific sequences in DNA. In plants with compound leaves as tomato (Solanum lycopersicum), KNOX I are expressed in the meristem, as well as on leaves, flowers and fruits, suggesting that they may play various roles in these organs. This hypothesis is supported by the intriguing phenotypes found in mutants with gain-of function of KNOX I genes, whose ectopic expression affects leaf, petal and fruit shape. An example, is the tomato mutant Mouse ear (Me), which overexpress the gene TKN2 (KNOX I). Similar phenotypes were also observed in transgenic plants overexpressing microRNA156 (miR156). MicroRNAs are a class of small no-coding RNAs (20-25 nucleotides) that are widely distributed in the genome of plants and animals, regulating the expression of their target genes by acting mainly at the post-transcriptional level. miR156 regulated post-transcriptionally most SQUAMOSA Promoter-Binding Protein-Like (SPL or SBP-box) genes, which encode plant-specific transcription factors. These genes play important roles in different aspects of development. To examine a possible molecular interaction between TKN2 transcription factor and microRNA156/SQUAMOSA Promoter-Binding Protein-Like module (miR156 node), it was evaluated the expression of miR156, its targets (SBP-box) and several genes downstream of miR156 node in different stages of the development of homozygous Me plants. Moreover, to evaluate the genetic interaction ...
Mestre

碩士
國立成功大學
熱帶植物科學研究所
99
MicroRNAs (miRNAs) are ~21-nucleotide noncoding RNAs that have been identified in various organisms. In plants, miRNAs are mostly involved in the regulation of important growth and developmental processes such as leaf and root development, phase transitions, and flowering. Recent studies explore two temporally expressed miRNAs in Arabidopsis, miR156 and miR172 play important roles in regulation of vegetative phase change and flowering time regulation. From juvenile-to-adult transition, the transcription of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) accumulation depend on the decrease of miR156 with time, and SPL genes promote miR172 transcript which then repress the APETALA2 (AP2), and AP2-like genes、TARGET OF EAT 1 (TOE1)、TOE2 to induce flowering. In this study, we used Phalaenopsis aphrodite subsp. formosana as material. To exam the possibility of controlling miR156 and miR172 expression in this long life cycle plant to accelerate the flowering time, we perform small RNA sequencing analysis and combining bioinformatics, we discovered 22 conserved miRNA families, and identified miR156 and miR172 othologs from Phalaenopsis orchid. Also, we discovered miR156 expresses higher in leaves than in stalks and in flower buds; however miR172 mainly expressed in stalks. These patterns are similar with those in Arabidopsis, imply us that we have possibility to overexpress miR172 to facilitate flowering in orchid the same way as Arabidopsis. Further, we identified the target genes of miR156 and miR172 from our orchid transcriptome databaes, PaSPL and PaAP2 which contain a miR156 and miR172 complementary site. The expression of PaSPL is abundant in stalks and flower buds that is complementary with expression of miR156. This suggests that miRNA-mediated flowering pathway exist in Phalaenopsis orchid.

碩士
國立成功大學
生命科學系
103
Organ size control of multicellular organisms poses a longstanding biological question that has always fascinated scientist. Especially leaf growth, by providing the food and renewable energy sources, is at the core of plant growth and undoubtedly an important part of human economic activities. Here, I report a novel Arabidopsis mutant, patronus1 (pans1), which reduced cell size and decreased cell number in leaves. The development of abaxial trichomes was also delayed in pans1. These facts indicate that pans1 mutant may accelerate heteroblasty, a phenomenon in which several traits of leaves change along with phase change, by alternating the cell number and size of cells, and trichome development. The genetic marker of leaf phase change, miR156, normally is down regulated in adult leaves but showed up-regulated in pans1. I also discovered PANS1 inhibits miR156 through its specific domains. Furthermore, miR156 downstream genes, SPL3, 5, 9 and10 had the decrease expression level in pans1 as a result of abnormal excessive miR156. Thus, I suggest that PANS1 plays an essential role of regulating vegetative phase change and promoting adult leaves growth in SPL-dependent pathway by inhibiting miR156 expression. Keywords: PANS1, phase change, heteroblasty, leaf development, miR156, SPL genes

碩士
國立中興大學
分子生物學研究所
107
Rice (Oryza Sativa L.) is an important crop in the world. As the climate changes and the population expand, there will be a shortage of food. Therefore, increasing food production is one of the important issues today. The number of tillers affect rice yield, so to screen rice with high tiller numbers and to study its possible molecular mechanism is the focus of this study. Two T-DNA activation-tagged mutants, M69217 and M11350, with high tiller numbers and dwarf phenotype were obtained from TRIM database. Both mutants showed enhanced expression of miR156, possibly due to the miR156 genes (Os-miR156d in M69217; Os-miR156b/c in M11350) flank to the inserted enhancer. Over-expression of Os-miR156d and Os-miR156b/c genes reproduced the mutant phenotype confirmed the function of miR156 genes. Although over-expression either Os-miR156d or Os-miR156b/c genes enhance tiller numbers, Os-miR156b/c enhance more tiller numbers than that of Os-miR156d. The stability or the secondary structure of the precursor might affect the maturation of miR156. To prove this hypothesis, the Os-miR156j with the most stable free energy counts and the Os-miR156a with the less stable free energy counts in the families were investigated. Current results showed the free energy differences in precursor of miR156 were not correlated to its effect on tiller numbers production. Strigolactone (SL) is a novel plant hormone involved in branching inhibition. The plants exhibit high branching (tiller in rice) and dwarf phenotype, when SL biosynthesis and its signal transduction genes is mutated. Expression of genes involved in SL biosynthesis and signal transduction were compared between WT and miR156 expression-enhanced rice plants. Results showed the SL biosynthesis gene Os-D10 and Os-SPL2 were reduced significantly in miR156 activation-tagged mutants and miR156 over-expression transgenic plants. To know how these two reduced genes are regulated by miR156 requires further investigations. To explore the function of individual Os-miR156 gene, a system using CRISPR/Cas9 to knock out miR156 gene with large deletion was established. With two target sites of CRISPR/Cas9 vector design; one out of 62 transgenic plants showed double cuts, resulted in a large deletion, to knock out miR156b/c gene was obtained. This system provides a way to study the function of miRNA genes and can be used to edit two target genes in one construct as well

碩士
國立臺灣大學
植物科學研究所
101
Previous studies have shown that high temperature-induced flowering is regulated by Oncidium cytosolic ascorbate peroxidase 1 (OgcytAPX1) through mediating the redox state of ascorbate (AsA) in Oncidium Gower Ramsey. Ecotopically overexpressing OgcytAPX1 in Arabidopsis can induce the expression of miRNA156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3) under high-temperature treatment. The miR156-SPL regulatory module has been known to regulate flowering in Arabidopsis, but its specific regulatory role on flowering is unclear. In order to dissect the function of miR156-SPL regulatory module on high temperature-induced flowering in Oncidium. We found three possible miR156-target SPL genes in Oncidium (OgSPLs). The study on expression profiling of Oncidium pseudobulbs at different growth stages indicated that miR156 steadily decreased, and OgSPLs increased as the Oncidium matures. Higher growth temperature increased OgSPLs transcription levels in both pseudobulbs and inflorescence buds, but the effect diminished after AsA application. However, the influence on miR156 under these treatments was not significant, suggesting that OgSPLs was independent of miR156 in high temperature-induced flowering mechanism. Meanwhile, ecotopically overexpressing OgSPLs in Arabidopsis mutant apx1 restored the delayed flowering phenotype compared to WT, suggesting that flowering regulation of OgSPLs was located at the downstream of OgcytAPX1. In conclusion, these results indicate that miR156 regulates phase transition through OgSPLs; however, OgSPLs act independently of age-mediated miR156 as one of the AsA redox downstream signal on the high temperature-induced flowering processes in the adult Oncidium.

Plant root architecture is regulated by the formation and function of meristems at the sites of root organogenesis. Meristem formation requires the generation of stem cell niches which produce the new cells of the growing organ. microRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root organogenesis. To identify key miRNAs involved in root meristem formation in M. truncatula, deep sequencing was used to compare the miRNA populations between root tip (RT), containing root apical meristem, and elongation zone (EZ) tissue plus root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. Functional analysis was performed on two of these predicted miRNAs, miRN304 and miR166g-5p. Over-expression of novel miRNA, miRN304, produces severe root abnormalities including stunted growth, loss of meristem structure and a poorly formed vasculature. mtr-miR166a-5p corresponds to the passenger strand of miR166a-3p, a miRNA previously shown to negatively regulate lateral root and nodule numbers. miR166a-5p over-expression results in increased nodule numbers and acts independently of miR166a-3p and may be a previously un-identified.