Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-09Bitstream added on 2014-06-13T20:54:10Z : No. of bitstreams: 1 moraes_fe_me_jabo.pdf: 1680634 bytes, checksum: 35809b1bab51b8d182c7d496667f751f (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cana-de-açúcar (Saccharum spp.) é uma das gramíneas cultivadas mais importantes do mundo e o Brasil é hoje o maior produtor e exportador mundial de cana, de açúcar de cana e etanol de cana, com o estado de São Paulo sendo o maior produtor. Entretanto, esta cultura é ameaçada por várias pragas e doenças. Dentre essas pragas, uma das mais importantes na região centro-sul é a cigarrinha-das-raízes (Mahanarva fimbriolata), que assumiu este posto devido ao aumento da colheita mecanizada. Por meio da técnica de cDNA-AFLP este trabalho teve como objetivo identificar genes possivelmente envolvidos na resistência a ninfas de Mahanarva fimbriolata em cana-de-açúcar. Para isso, foi utilizada uma variedade suscetível (SP80-1816) e uma resistente (SP83-5073). Ambas foram infestadas com ninfas da cigarrinha 77 dias após o plantio e amostras de raízes foram coletadas antes da infestação (controle) e 1, 2 e 7 dias após a infestação, posteriormente foi feita a extração de RNA, síntese de cDNA e o cDNA-AFLP. Após a análise dos fragmentos foram encontrados 16 fragmentos expressos somente na variedade resistente. Nove fragmentos foram encontrados no 2º dia de infestação e sete no 7º dia. Dentre os 16 fragmentos, sete foram recuperados e sequenciados. Seis fragmentos não apresentaram homologia com proteínas já descritas e um fragmento, de 129 pb, apresentou homologia com uma proteína putativa associada à senescência. Todos os fragmentos podem representar genes relacionados com a resistência da planta contra o inseto / The sugarcane (Saccharum spp.) is the most important grass cultivated in the world and Brazil is now the world’s largest producer and exporter of sugarcane, sugarcane sugar and sugarcane ethanol, with the São Paulo state being the largest producer. However, this culture is threatened by several pests and diseases. Among these pests, one of the most important in south-central region is the froghopper-roots (Mahanarva fimbriolata), that took this position because of increased mechanization. Through cDNA-AFLP technique this study aimed to identify genes possibly involved in resistance to nymphs of Mahanarva fimbriolata in sugarcane. Therefore we used a susceptible variety (SP80-1816) and a resistant (SP83-5073). Both were infested with spitlebugs 77 days after planting and root samples were collected before infestation (control) and 1, 2 and 7 days after infestation, was later made the extraction of RNA, cDNA synthesis and cDNA-AFLP. After fragments analyses were found 16 fragments expressed only in the resistant variety. Nine fragments were found on day 2 of infection and seven on day 7. Among the 16 fragments, seven were recovered and sequenced. Six fragments showed no homology to described proteins and a fragment of 129 bp showed homology to a putative protein associated with senescence. All fragments could represent genes related to the plant resistance against the insect

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No. of bitstreams: 1 melloni_mlg_dr_jabo_par.pdf: 733459 bytes, checksum: d8b36f206c9b78d184a6e2627f86b26d (MD5) Previous issue date: 2015-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Com o intuito de aumentar o conhecimento da rede gênica envolvida no controle do florescimento em cana-de-açúcar, diferentes cultivares de cana-de-açúcar foram submetidos a tratamentos fotoperiódicos de indução e não indução do florescimento em câmara de fotoperíodo. Aos 5, 10 e 20 dias de indução, a folha +1 e a bainha foliar foram coletadas para a identificação de fragmentos diferencialmente expressos (FDEs) por meio da técnica de cDNA-AFLP entre e dentro dos tratamentos fotoperiódicos. Um total de 162 fragmentos foram selecionados e reamplificados. Destes, 63 FDEs tiveram sucesso na reação de reamplificação e foram clonados e sequenciados. As sequências foram confrontadas com seis bancos de sequências: 1. Transcritos do projeto SUCEST ;2. Proteínas do genoma de sorgo; 3. BAC de cana-de-açúcar; 4. Proteínas do genoma de arroz, 5. Proteínas presentes no Phytozome e 6. NCBI. A busca por similaridade se deu pelo uso da ferramenta BLASTn (e-value 1e-5) nos casos do banco SUCEST e dos BACs de cana-de-açúcar e BLASTx (e-value 1e-5) para os demais bancos. Dentre os 63 FDEs, 23 corresponderam a sequências de genes enquanto os outros 40 representam sequências que não estão depositadas nestes bancos (no hits). A maioria das 23 sequencias apresenta similaridade com genes que codificam proteínas hipotéticas ou preditas em diversos organismos. Com base na análise do domínio da proteína realizada pelo Pfam, seis sequências podem estar associadas ao metabolismo da indução do florescimento. Dentre estas as sequencias LM-19, LM-40 e LM-53 se destacaram. A LM-19 possui similaridade com o gene que codifica uma proteína com o domínio DNAJ sendo que proteína com este domínio é considerada mediador da integração dos sinais do florescimento em Arabidopsis thaliana. LM-40 possui similaridade com o gene que codifica proteína de domínio (F-BOX); estudos indicam forte relação deste domínio aos processos de indução ao florescimento. LM-53 tem um domínio de proteína predita semelhante ao domínio da proteína codificada pelo gene CONSTANS que regula a expressão de FLOWERING LOCUS T (FT), que codifica o florígeno em Arabidopsis thaliana e em algumas outras espécies. De maneira geral, a técnica do cDNA-AFLP foi eficiente, na identificação de FDEs ao longo dos tratamentos fotoperiódicos de indução e não indução do florescimento. Os resultados obtidos sugerem que as sequencias LM-19, LM-40 e LM-53 estão vinculadas aos metabolismos de indução do florescimento. É provável que a maioria dos FDEs obtidos possam estar envolvidos nos metabolismos da indução do florescimento, porém ainda não foram identificados na literatura. / In order to increase the knowledge of the gene network involved in sugarcane flowering induction, sugarcane cultivars were submitted to different photoperiod treatments of flowering induction and non-induction in a photoperiod facility. At 5, 10 and 20 days of induction, the +1 leaf and the leaf sheath were collected for the identification of different transcript-derived fragments (TDFs) within and between the photoperiod treatments to apply the cDNA-AFLP technique. A total of 162 TDFs were selected and re-amplified. Of these, 63 TDFs were successful in re-amplification and were cloned and sequenced. The sequences were confronted against 6 sequence databanks (SUCEST transcripts; Sorghum genome proteins; Sugarcane BACs; proteins from rice genome; Phytozome and NCBI). Similarity search was done by using the BLASTn (e-value 1e-5) tool for the SUCEST databank and sugarcane BACs while BLASTx (e-value 1e-5) was use for the other banks. Among the 63 TDFs, 23 corresponded to gene sequences while the remaining 40 represent sequences that are not deposited in these banks (no hits). The majority of the 23 sequences showed similarity with genes coding for hypothetical or predicted proteins of different organisms. Based on the protein domain analysis conducted by Pfam, six sequences may be associated with flowering induction metabolism. Among these: LM-19, LM-40 and LM-53 sequences stood out. LM-19 has similarity to the gene encoding a protein with DnaJ domain. Proteins having this domain are considered as an integrating floral signals mediator in Arabidopsis thaliana. LM-40 has similarity to the gene encoding a protein with (F-BOX) domain. This domain has a strong relationship in flowering induction processes. LM-53 has one of the predicted protein domain similar to the domain of the protein encoded by the CONSTANS gene which governs the expression of FLOWERING LOCUS T (FT), this later one encodes the florigen. Generally the cDNA-AFLP technique was effective in identifying TDFs across the flowering inductive and non-inductive photoperiodic treatments. The results suggest that LM-19, LM-40 and LM-53 sequences are linked to flowering induction metabolisms. Probably, most of the TDF here obtained may be involved in the flowering induction metabolism, although not yet been identified in the literature. / FAPESP: 2013/24020-0

This study represents the first comprehensive analysis of gene regulation underlying the distinct categories of resistance afforded to wheat (Triticum aestivum, L.) by different Dn genes. Russian wheat aphid (Diuraphis noxia, Mordv.) feeding on susceptible wheat cultivars causes leaf rolling, chlorosis and the eventual death of the plant. Plants expressing Dn genes are resistant to D. noxia infestation, but different Dn genes afford phenotypically distinct modes of resistance: the Dn1 gene confers an antibiotic effect to lower aphid fecundity; Dn2 confers tolerance to high aphid pressure; and Dn5 confers antixenosis, and aphids do not prefer such plants as hosts. Little is known about the components involved in establishing a successful defence response against D. noxia attack and how these differ between the distinct resistance categories. It is assumed that the Dn genes function as classic R genes in plant defence, being receptors for elicitors in aphid saliva. Upon recognition, defence response signalling is initiated, but the exact mechanics of subsequent cellular events in aphid resistance have only recently come under investigation. Evidence from cDNA microarray and subtractive hybridization experiments indicated the involvement of kinase signalling cascades and photosynthetic proteins in the response against D. noxia. However, expression analysis describing how these processes differ between plants carrying different Dn genes and how these differences account for antibiosis, antixenosis or tolerance had not been conducted. We consequently investigated the downstream components involved in or affected by the generation of these resistance mechanisms by comparing the responses in transcript regulation of Tugela near-isogenic lines with different Dn genes to D. noxia infestation. cDNA-AFLP analysis was selected as an appropriate functional genomics tool, since it is semi-quantitative, does not require prior sequence information and allows for the discovery of novel genes. cDNA-AFLP analysis yielded 121 differentially regulated transcript-derived fragments (TDFs) grouped into eight expression clusters. We cloned and sequenced 49 representative TDFs, which were further classified into five broad functional categories based on inferred similarity to database sequences. Transcripts involved in such diverse processes as stress, signal transduction, photosynthesis, metabolism and gene regulation were found to be differentially regulated during D. noxia feeding. Many TDFs demonstrated homology to proteins with unknown function and several novel transcripts with no similarity to previously published sequences were also discovered. Detailed expression analysis using quantitative RT-PCR and RNA hybridization provided evidence that the time and intensity of induction of specific pathways is critical for the development of a particular mode of resistance. This includes: the generation of kinase signalling cascades and the induction of several ancillary processes such as ubiquitination, leading to a sustained oxidative burst and the hypersensitive response during antibiosis; tolerance as a passive resistance mechanism countering aphid-induced symptoms through the repair or de novo synthesis of photosystem proteins; and the possible involvement of ethylene-mediated wounding pathways in generating volatile organic compounds during antixenosis. This is the first report on the involvement of KCO1, a vacuolar K+ channel, in assisting cytosolic Ca2+-influx and preventing leaf rolling, as well as on the role of iron homeostasis as a gene regulatory mechanism for sustaining the oxidative burst during the antibiotic defence response. This study opens up several areas of investigation heretofore unexplored in cereal-aphid interaction research. Of particular interest is the induction of genes involved in photosynthetic compensation during Dn2 tolerance responses, since these constitute a novel, passive resistance mechanism exclusive to aphid defence as opposed to the active resistance triggered in the presence of the Dn1 gene in the form of a general hypersensitive response. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

La conductivité hydraulique foliaire (KF) est une composante majeure du transport d'eau dans toute la plante. Dans les feuilles de noyer, la KF est stimulée à la lumière et est étroitement liée à l'accroissement du taux des transcrits d'aquaporines JrPIP2s. Par ailleurs, la corrélation entre la stimulation de la KF et des transcrits d'aquaporines à la lumière, n'est pas générale et dépend de l'espèce. Ici, nous étudions cette corrélation chez cinq espèces forestières (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba et Populus tremula) différant par leur réponse à la lumière. Nous démontrons seulement chez le noyer (Juglans regia), la contribution des deux familles d'aquaporines PIP1s et PIP2s. Afin de mieux comprendre le rôle des JrPIP1s et JrPIP2 dans la réponse à la lumière, nous avons isolé 8 nouvelles isoformes dans les feuilles de noyer et nous avons étudié leurs profils d'expression sur une cinétique lumière. Toutes les isoformes étudiées sont accumulées à la lumière et réprimées à l'obscurité. De plus, la KF est dépendante de la qualité de lumière. Elle est réduite de 65% en absence de lumière bleue. Cette diminution serait liée à l'inhibition des transcrits d'aquaporines. Afin de caractériser les mécanismes moléculaires précoces impliqués dans la modulation de KF par la lumière, l'approche globale cDNA-AFLP a été menée sur des feuilles de noyer sous différentes conditions d'éclairement. Nous obtenons 12000 transcrits différentiels dérivés (TDFs) générés par les 128 couples d'amorces. Parmi les 187 séquences obtenues, 93 d'entre elles ont une fonction putative. Leur classification fonctionnelle montre que les gènes relatifs à la régulation cellulaire représentent environ 58% des TDFs identifiés. Les feuilles exposées à la lumière, montrent des changements dans les voies de : signalisation calcique, protéolyse, trafic vésiculaire et l'expression de divers facteurs de transcription et protéines de régulation. Pour mieux comprendre le rôle potentiel de la signalisation calcique dans la modulation de la KF par la lumière, nous avons étudié l'effet d'un inhibiteur des canaux calciques [LaCl3] et d'un antagoniste de calmoduline [W7] sur la KF et les transcrits des 10 JrPIPs. Comparées aux feuilles témoins, les inhibiteurs calciques provoquent une réduction de la KF et de la majorité des JrPIPs étudiées à la lumière. Nos résultats confirment l'implication du complexe Ca2+ /calmoduline dans la transduction du signal lumineux responsable de la stimulation de la KF et des transcrits d'aquaporines chez le noyer.