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No. of bitstreams: 1 Tese - Marcicleide Lima do Espirito Santo.pdf: 7339955 bytes, checksum: 22519cc9c5e5f14e2991f1e20ddb938f (MD5) Previous issue date: 2006-06-22 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / The camu-camu (Myrciaria dubia (H.B.K.) McVaugh) is a native sort of the Amazonian region, whose fruit presents elevated content of ascorbic acid (vitamin C). The study of the functional genome in camu-camu fruits has like base the expressed sequence tags sequencing - ESTs. Faced with the displayed the present thesis is going to analyze and identify express genes in camu-camu fruits by means of ESTs sequencing. The total RNA was extracted from the shell-pulp. The extremity 5’sequencing' of cDNA insert was carried out so much in the Technology of the DNA Laboratory (UFAM) and in the Sequencing Platform (EMBRAPA/CENARGEN). The ESTs sequences obtained were submitted to the System Genome, program of genomic annotation that integrates analysis management programs and viewing of nucleotides sequences. It developed an efficient procedure for total RNA extraction of camu-camu fruits that enabled the obtaining of mRNAs of quality, utilized in the making of cDNAs of sizes varied (500pb to 4Kb). From the sequencing were obtained 3196 ESTs valid, being formed 1546 singletons and 358 contigs, resulting of 2586 ESTs sequences in total with similarity the sequences found in the gene bank. The analysis library clusterization revealed an index of 81% novelty and 32,54% redundancy. Around 90% of the contigs presented decrease redundancy (2-4 reads by contigs). The facts of the categorization of the proteins identified detached the posttranslational modification, protein turnover, chaperones (13,2%) category. From the hoist of the species with bigger number of ESTs with similarity the camu-camu sequences detached itself Arabidopsis thaliana with 49%. Around 10 uniques presented very high similarity (and-value 0.0) to known genes. The ESTs more abundantly express in camu-camu fruits encode to gluthatione s-transferase. They were observed around 3% sequences (97 ESTs) with decrease similarity (e-value > e-10) and 15% did not they present similarity with no contained sequence in the gene bank. They were identified 138 ESTs sequences (4,3%) that they encode molecular chaperones with prevalence of the sHSP family that represents 33% of express chaperones. ESTs related to the ascorbic acid metabolism also were identified, being nine related the synthesis and six come back for ascorbic acid conversion and recycling. ESTs related to the ripening and mechanisms of defense of the fruit also were noticeable. / O camu-camu (Myrciaria dúbia (H.B.K.) McVaugh) é uma espécie nativa da região Amazônica, cujo fruto apresenta elevado teor de ácido ascórbico (vitamina C). O estudo do genoma funcional em frutos de camu-camu tem como base o seqüenciamento de fragmentos de seqüências expressas - ESTs (Expressed Sequence Tags). Diante do exposto a presente tese pretende analisar e identificar genes expressos em frutos de camu-camu por meio de seqüenciamento de ESTs. O RNA total foi extraído a partir da casca-polpa. O seqüenciamento da extremidade 5’ de insertos de cDNA foi realizado tanto no Laboratório de Tecnologia do DNA da UFAM e como na Plataforma de Seqüenciamento da EMBRAPA/CENARGEN. As seqüências ESTs obtidas foram submetidas ao Sistema Genoma, programa de anotação genômica que integra programas de gerenciamento de análise e visualização de seqüências nucleotídicas. Os resultados obtidos foram o desenvolvimento de um procedimento eficiente para extração de RNA total de frutos de camu-camu que possibilitou a obtenção de mRNAs de qualidade, utilizados na confecção de cDNAs de tamanhos variados (500pb a 4Kb). A partir do seqüenciamento foram obtidas 3196 ESTs válidas, sendo formados 1546 singletons e 358 contigs, resultando num total de 2586 seqüências ESTs com similaridade a seqüências encontradas no banco de genes. A análise da clusterização da biblioteca revelou um índice de 81% de novidade e 33% de redundância. Cerca de 90% dos contigs apresentaram baixa redundância (2-4 reads por contigs). Os dados da categorização das proteínas identificadas destacaram a categoria modificação pós-traducional, proteína turnover, chaperonas (13,2%). A partir do levantamento das espécies com maior número de ESTs com similaridade a seqüências de camu-camu destacou-se Arabidopsis thaliana com 49%. Cerca de 10 uniques apresentaram altíssima similaridade (e-value 0.0) a genes conhecidos. Os ESTs mais abundantemente expresso em frutos de camu-camu codificam a glutationa s-transferase. Foram observados cerca de 3% de seqüências (97 ESTs) com baixa similaridade (e-value > e-1010) e 15% não apresentaram similaridade com nenhuma seqüência contida no banco de genes. Foram identificadas 138 seqüências ESTs (4,3%) que codificam chaperonas moleculares com destaque à família sHSP que representa 33% das chaperonas expressas. ESTs relacionados ao metabolismo do ácido ascórbico também foram identificados, sendo nove relacionados a síntese e seis voltados para conversão e reciclagem do ácido ascórbico. ESTs relacionados ao amadurecimento e mecanismos de defesa do fruto também foram destacados.

Resumo: Sob condições de estresse hídrico, a cana-de-açúcar pode sofrer mudanças fisiológicas e bioquímicas, tais como diminuição nas atividades fotoquímicas, redução da fixação de CO2 e acúmulo de osmólitos e osmoprotetores. O objetivo deste trabalho foi identificar, através da técnica de macroarranjo de cDNA, o perfil de expressão de genes promotores das diferentes vias metabólicas em palmitos da variedade de cana-de-açúcar SP80-3280 submetidas ao estresse hídrico nos dias 5, 9, 13 e 17 após o início da condição de supressão de água, sendo considerado o dia 1 como controle. Os resultados do macroarranjo mostraram que as proteínas mais expressas sob déficit hídrico pertencem a quatro categorias das quais as ESTs mais importantes foram selecionadas. As quatro categorias descritas abaixo estão discutidas neste trabalho. As ESTs da via do metabolismo de açúcar e amido (invertase de parede celular (INV), sacarose fosfato sintase (SFS), sacarose fosfato fosfatase (SFF), trealose fosfato sintase (TFS), trealose fosfato sintase/fosfatase (TFS/F) e hexoquinase (HXQ)) pertencem a categoria de bioenergética. Colina monooxigenase (CMO) e betaína aldeído desidrogenase (BADH) as quais pertencem a via do metabolismo de glicina betaína, foram selecionadas a partir da categoria do metabolismo secundário. A terceira categoria compreende as ESTs do metabolismo de aminoácido (D-pirrolina-5- carboxilato sintase (P5CS), ornithina -aminotransferase (OAT) e prolil 4-hidroxilase (PH)) as quais pertencem a via da biossíntese de prolina, e a quarta categoria, resposta ao estresse, compreende as ESTs aleno oxido sintase (AOS), aleno oxido ciclase (AOC) e lipoxigenase (LOX), pertencentes a via de biossíntese do jasmonato. / Abstract: Under water deficit, sugarcane undergoes physiological and biochemical alterations such as photochemical activity reduction, CO2 fixation reduction and the accumulation of osmolytes and osmoprotectants. This work was undertaken to identify the gene expression profile in sugarcane (var. SP80-3280) under water deficit through the cDNA macroarray technique. Leafroll tissues were collected from plants subjected to 5, 9, 13 and 17 days of water restriction, and day 1 was used as control. Macroarray results showed that most proteins expressed under water restriction belong to four categories, from which the most important ESTs were selected. The four categories described below are discussed in this work. Sugar and starch metabolism pathway ESTs (cell wall invertase (CWI), sucrose phosphate synthase (SPS), sucrose phosphate phosphatase (SPP), trehalose phosphate synthase (TPS), trehalose phosphate synthase phosphatase (TSP/P) and hexoquinase (HXQ) were selected from the bioenergetics category. Choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), which belong to the glycine betaine metabolism pathway, were selected from the secondary metabolism category. The third category comprised the amino acid metabolism ESTs D- pyrroline-5-carboxylate synthase (P5CS), Ornithine - aminotransferase (OAT) and Prolyl 4-hydroxylase (PH), which belong to the proline biosynthesis pathway, and the fourth category, stress response, comprised the ESTs for Allene oxide synthase (AOS), Allene oxide cyclase (AOC) and Lipoxygenase (LOX), which belong to the jasmonate biosynthesis pathway. / Orientador: Maria Inês Tiraboschi Ferro / Coorientador: Sonia Marli Zingaretti / Coorientador: Roberto Willians Noda / Banca: João Suzuki / Banca: Poliana Fernanda Giachetto / Mestre

Doctor of Philosophy / Department of Entomology / C. M. Smith / Brenda Oppert / Coleopteran insects compensate for dietary protease inhibitors by a number of mechanisms. To study this compensation response at the molecular level, the digestive proteases of Tenebrio molitor were studied. Biochemical studies of the pH optima and inhibitor sensitivity of proteases indicated the cysteine proteases were mostly in the anterior and serine proteases were in the posterior midgut of T. molitor larvae. Expressed Sequence Tags (ESTs) from T. molitor larval midgut cDNA libraries contained sequences encoding putative digestive proteases. Of a total of 1,528 cDNA sequences, 92 cDNAs encoded proteases, and 50 full-length cDNAs were grouped into serine, cysteine and metallo protease classes. Sequences tmt1a, tmt1b and tmt1c were identified as genes encoding isoforms of T. molitor trypsin, and tmc1a encoded T. molitor chymotrypsin. The general distribution cysteine protease transcripts in the anterior and serine protease transcripts in the posterior midgut, of T. molitor larvae, was in agreement with the biochemically-characterized compartmentalization of proteases. Expression analyses of selected transcripts demonstrated varied expression patterns across five developmental stages of T. molitor, with maximal expression of most protease transcripts in first instar larvae. Dietary serine and cysteine protease inhibitors fed in combination to early-instar T. molitor larvae caused a significant delay in larval growth in 21-day-old larvae. Real-time quantitative PCR analysis of RNA isolated from larvae fed different protease inhibitor treatments indicated that dietary inhibitors affected the expression of serine and cysteine proteases. Larvae fed soybean trypsin inhibitor, a serine protease inhibitor, compensated by the hyperproduction of proteases from the same class, as well as the upregulation of cysteine proteases. A cysteine protease inhibitor, E-64, caused a reduction in the hyperproduction of all proteases, and, in combination with the soybean trypsin inhibitor, lowered the compensation response of T. molitor larvae to negligible levels. These data suggest that T. molitor larvae are more sensitive to the effects of cysteine protease inhibitors, perhaps because these proteases are the first line of defense for larvae against plant protease inhibitor. The bioassay and molecular studies suggested that combinations of inhibitors that target both serine and cysteine proteases are needed to effectively control larval infestations of T. molitor.

Made available in DSpace on 2014-06-11T19:26:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-20Bitstream added on 2014-06-13T18:54:09Z : No. of bitstreams: 1 jovino_dfr_me_jabo.pdf: 637580 bytes, checksum: dc449a2a52a9354da867effed1512963 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Sob condições de estresse hídrico, a cana-de-açúcar pode sofrer mudanças fisiológicas e bioquímicas, tais como diminuição nas atividades fotoquímicas, redução da fixação de CO2 e acúmulo de osmólitos e osmoprotetores. O objetivo deste trabalho foi identificar, através da técnica de macroarranjo de cDNA, o perfil de expressão de genes promotores das diferentes vias metabólicas em palmitos da variedade de cana-de-açúcar SP80-3280 submetidas ao estresse hídrico nos dias 5, 9, 13 e 17 após o início da condição de supressão de água, sendo considerado o dia 1 como controle. Os resultados do macroarranjo mostraram que as proteínas mais expressas sob déficit hídrico pertencem a quatro categorias das quais as ESTs mais importantes foram selecionadas. As quatro categorias descritas abaixo estão discutidas neste trabalho. As ESTs da via do metabolismo de açúcar e amido (invertase de parede celular (INV), sacarose fosfato sintase (SFS), sacarose fosfato fosfatase (SFF), trealose fosfato sintase (TFS), trealose fosfato sintase/fosfatase (TFS/F) e hexoquinase (HXQ)) pertencem a categoria de bioenergética. Colina monooxigenase (CMO) e betaína aldeído desidrogenase (BADH) as quais pertencem a via do metabolismo de glicina betaína, foram selecionadas a partir da categoria do metabolismo secundário. A terceira categoria compreende as ESTs do metabolismo de aminoácido (D-pirrolina-5- carboxilato sintase (P5CS), ornithina -aminotransferase (OAT) e prolil 4-hidroxilase (PH)) as quais pertencem a via da biossíntese de prolina, e a quarta categoria, resposta ao estresse, compreende as ESTs aleno oxido sintase (AOS), aleno oxido ciclase (AOC) e lipoxigenase (LOX), pertencentes a via de biossíntese do jasmonato. / Under water deficit, sugarcane undergoes physiological and biochemical alterations such as photochemical activity reduction, CO2 fixation reduction and the accumulation of osmolytes and osmoprotectants. This work was undertaken to identify the gene expression profile in sugarcane (var. SP80-3280) under water deficit through the cDNA macroarray technique. Leafroll tissues were collected from plants subjected to 5, 9, 13 and 17 days of water restriction, and day 1 was used as control. Macroarray results showed that most proteins expressed under water restriction belong to four categories, from which the most important ESTs were selected. The four categories described below are discussed in this work. Sugar and starch metabolism pathway ESTs (cell wall invertase (CWI), sucrose phosphate synthase (SPS), sucrose phosphate phosphatase (SPP), trehalose phosphate synthase (TPS), trehalose phosphate synthase phosphatase (TSP/P) and hexoquinase (HXQ) were selected from the bioenergetics category. Choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), which belong to the glycine betaine metabolism pathway, were selected from the secondary metabolism category. The third category comprised the amino acid metabolism ESTs D- pyrroline-5-carboxylate synthase (P5CS), Ornithine - aminotransferase (OAT) and Prolyl 4-hydroxylase (PH), which belong to the proline biosynthesis pathway, and the fourth category, stress response, comprised the ESTs for Allene oxide synthase (AOS), Allene oxide cyclase (AOC) and Lipoxygenase (LOX), which belong to the jasmonate biosynthesis pathway.

Leaf rust caused by Puccinia hordei is an important disease of barley (Hordeum vulgare) in many regions of the world. Yield losses up to 62% have been reported in susceptible cultivars. The Rph5 gene confers resistance to the most prevalent races (8 and 30) of barley leaf rust in the United States. Therefore, the molecular mapping of Rph5 is of great interest. Genetic studies were performed by analysis of 93 and 91 F2 plants derived from the crosses 'Bowman' (rph5) x 'Magnif 102' (Rph5) and 'Moore' (rph5) x Virginia 92-42-46 (Rph5), respectively. Linkage analysis positioned the Rph5 locus to the extreme telomeric region of the short arm of barley chromosome 3H at 0.2 cM proximal to RFLP marker VT1 and 0.5 cM distal from RFLP marker C970 in the Bowman x Magnif 102 population. Synteny between rice chromosome 1 and barley chromosome 3 was employed to saturate the region within the sub-centimorgan region around Rph5 using sequence-tagged site (STS) markers that were developed based on barley expressed sequence tags (ESTs) syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising distal region of the rice chromosome 1S. Five rice PAC clones were used as queries to blastn 370,258 barley ESTs. Ninety four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 10 EST-based STS markers were incorporated into the 'Bowman' x 'Magnif 102' high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, co-segregate with Rph5. Genes, represented by these markers, are putative candidates for Rph5. Results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley EST resources for marker saturation of targeted barley genomic region. / Ph. D.