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Caracterização genética e análise evolutiva In silico de aquaporinas em feijão-caupi (Vigna unguiculata (L.) Walp.)BEZERRA NETO, João Pacífico 31 January 2012 (has links)
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Previous issue date: 2012 / CAPES; CNPQ; FACEPE; BNB; FINEP / O feijão-caupi é uma das mais antigas leguminosas cultivadas, com destacada
importância em áreas tropicais e subtropicais. No Brasil, é uma das principais fontes
de nutrientes para a dieta humana e animal, representando 80% da produção de
grãos nas regiões norte e nordeste. Entretanto, fatores como seca e salinidade (as
duas principais adversidades ambientais em regiões semiáridas) têm prejudicado
tanto a quantidade quanto a qualidade da produção do feijão-caupi. Neste sentido, o
estudo de proteínas que agem como facilitadoras da passagem de água entre as
membranas biológicas e na prevenção da passagem de íons e outros solutos na
célula é fundamental para o entendimento de como algumas plantas respondem a
tais adversidades. Entre estas proteínas as aquaporinas merecem atenção, tratandose
de uma família de pequenas proteínas transmembranas (24-30 kDa), dividida em
quatro subfamílias, incluindo as Proteínas de Membrana Plasmática (PIP), Proteínas
de Membrana de Tonoplasto (TIP), Proteínas de Membrana de Nódulos (NIP) e
Pequenas Proteínas Básicas Intrínsecas de Membrana (SIP). Apesar da grande
quantidade de dados sobre aquaporinas nos vegetais, nada se sabe sobre estes
transportadores em algumas culturas de importância econômica, como o feijãocaupi.
Utilizando dados de EST (Expressed Sequence Tags), 37 transcritos
candidatos a aquaporinas foram identificados em feijão-caupi por meio de buscas
com ferramentas de bioinformática, onde 27 apresentavam o domínio íntegro e 10
encontravam-se incompletas. Análises fenéticas das sequências de aminoácidos
dividiram esta família nas quatro subfamílias já conhecidas para outras espécies
vegetais, sendo 11 PIPs (cinco PIP1 e seis PIP2), 10 TIPs (quatro TIP3 e seis TIP),
quatro NIPs e duas SIPs. Os alinhamentos múltiplos gerados a partir das sequências
com domínio MIP completo, indicaram que as regiões ar/R das aquaporinas do
feijão-caupi, em sua maioria, são similares às presentes em Arabidopsis, milho e
arroz, apontando para afinidades com o mesmo tipo de soluto transportado. A
expressão dos transcritos foi observada em todos os tecidos vegetais, estando
associadas ao transporte de diferentes tipos de solutos nos compartimentos
celulares. As aquaporinas identificadas neste estudo representam um importante
recurso genético, fornecendo alvos em potencial para modificar as propriedades do
uso de água em feijão-caupi ou em outras leguminosas relacionadas.
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Genomic, expression and functional analysis of genes from larval gut of the European corn borer, Ostrinia nubilalis (Hübner)Khajuria, Chitvan January 1900 (has links)
Doctor of Philosophy / Department of Entomology / Larry L. Buschman / Kun Yan Zhu / Genomic information for lepidopteran insects, particularly agricultural pest species, is very limited but urgently needed due to their economic importance and biodiversity. The huge economic losses ($ 1-2 billons / year) caused by the European corn borer (Ostrinia nubilalis, Hübner, ECB) makes this insect species one of the major pests of corn in the United States and western world. Management of ECB by conventional methods is limited but has had a great success by transgenic Bt (Bacillus thuringiensis) corn, which targets insect gut. However, the widespread use of Bt corn may lead to the development of Bt resistance in ECB. Knowledge of genes expressed in the insect gut is considered crucial for understanding basic physiology of food digestion, their interactions with Bt toxins and pathogens, and for discovering new targets for pest management.
A large database of 15,000 expressed sequence tags (ESTs) was established from the ECB larval gut. To our knowledge, this database represents the largest gut-specific EST database from a lepidopteran pest. Analysis of 10 aminopeptidase-like genes between Cry1Ab–resistant and –susceptible ECB larvae revealed that aminopeptidase P-like (OnAPP) gene is a strong candidate for its role in Bt toxicity and resistance. The RNA interference mediated reduction in the transcript level of OnAPP gene in ECB larvae resulted in their reduced susceptibily to Cry1Ab.
Analysis of the chitinase-like gene (OnCht) revealed its essential role in regulating chitin content of peritrophic membrane (PM). Our results suggest that OnCht may influence food digestion, nutrient absorption or movement of digestive enzymes through the PM and can be an important target for insect management. We also identified and characterized six genes involved in the innate immune defense response in ECB and showed that the expression of these genes were induced when challenged with bacteria.
In addition to these results, this research generated significant genomic information for the development of microarray from the larval gut of ECB. The establishment of the feeding-based RNA interference technique could potentially help in delivering dsRNA orally to ECB for high throughput screening of effective genes to be targeted for insect pest management.
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Recherche statistique de biomarqueurs du cancer et de l'allergie à l'arachide / Development of statistical methods for the discovery of novel biomarkers for cancer or peanut allergyCollignon, Olivier 16 October 2009 (has links)
La première partie de la thèse traite de la recherche de biomarqueurs du cancer. Lors de la transcription, il apparaît que certains nucléotides peuvent être remplacés par un autre nucléotide. On s'intéresse alors à la comparaison des probabilités de survenue de ces infidélités de transcription dans des ARNm cancéreux et dans des ARNm sains. Pour cela, une procédure de tests multiples menée sur les positions des séquences de référence de 17 gènes est réalisée via les EST (Expressed Sequence Tag). On constate alors que ces erreurs de transcription sont majoritairement plus fréquentes dans les tissus cancéreux que dans les tissus sains. Ce phénomène conduirait ainsi à la production de protéines dites aberrantes, dont la mesure permettrait par la suite de détecter les patients atteints de formes précoces de cancer. La deuxième partie de la thèse s'attache à l'étude de l'allergie à l'arachide. Afin de diagnostiquer l'allergie à l'arachide et de mesurer la sévérité des symptômes, un TPO (Test de Provocation Orale) est réalisé en clinique. Le protocole consiste à faire ingérer des doses croissantes d'arachide au patient jusqu'à l'apparition de symptômes objectifs. Le TPO pouvant se révéler dangereux pour le patient, des analyses discriminantes de l'allergie à l'arachide, du score du TPO, du score du premier accident et de la dose réactogène sont menées à partir d'un échantillon de 243 patients, recrutés dans deux centres différents, et sur lesquels sont mesurés 6 dosages immunologiques et 30 tests cutanés. Les facteurs issus d'une Analyse Factorielle Multiple sont également utilisés comme prédicteurs. De plus, un algorithme regroupant simultanément en classes des intervalles comprenant les doses réactogènes et sélectionnant des variables explicatives est proposé, afin de mettre ensuite en compétition des règles de classement. La principale conclusion de cette étude est que les mesures de certains anticorps peuvent apporter de l'information sur l'allergie à l'arachide et sa sévérité, en particulier ceux dirigés contre rAra-h1, rAra-h2 et rAra-h3. / The first part of this doctoral dissertation deals with the research of cancer biomarkers. During transcription it was observed that some nucleotides are replaced mistakenly by others. We sought to compare the probabilities of these transcription infidelities in mRNA originating from normal and cancerous tissues. To do this, a multiple testing procedure was performed on the positions of 17 genes by considering their ESTs (Expressed Sequence Tag). The conclusion was reached that the proportions of these transcription errors are mainly increased in cancer tissues as compared to normal ones. This phenomenon would lead to the translation of aberrant proteins, whose detection could help in identifying patients with cancer. The main goals of the second part are the diagnosis of peanut allergy and the prediction of its severity. Diagnosing peanut allergy and evaluating the intensity of the symptoms are currently accomplished with a double blind placebo controlled food challenge (DBPCFC). Patients are given increasing peanut doses until the first clinical reaction appears. Since DBPCFC can result in life-threatening responses, we propose an alternate procedure with the long term goal of replacing invasive allergy tests. Discriminant analyses of peanut allergy, DBPCFC score, the eliciting dose and the first accidental exposure score were performed in 243 allergic patients using 6 immunoassays and 30 skin prick tests. A Multiple Factorial Analysis was performed to use new factors as predictors. We also developed an algorithm for simultaneously clustering eliciting dose values and selecting discriminant variables. Our main conclusion is that antibody measurements provide information on the allergy and its severity, especially those directed against the peanut allergens rAra-h1, rAra-h2 and rAra-h3.
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Short sequence tags reveal global transcription of repetitive elements in mammalian genomesGeoffrey Faulkner Unknown Date (has links)
Retrotransposon mobilization is a major source of genome evolution. However, the functional consequences of these events, and particularly their influence upon transcriptional activity, are poorly defined. The extent of retrotransposon transcription, as well as that of other repetitive elements, has eluded systematic study due to difficulties in discriminating elements copied in multiple genomic loci. Moreover, the potential regulatory effects of retrotransposon transcription upon the expression of neighbouring protein-coding genes are also largely unknown. This thesis develops methods to survey repetitive element expression and assess the functions of retrotransposons in the mouse and human genomes. Chapter 1 summarises the complex transcriptional output of the mammalian genome, the functional annotation of this expression and the genomic and bioinformatic tools available for its detection. Chapter 2 explores the capacity of short sequence tags to discern transcription from individual repetitive elements, as well as from protein-coding genes. It is based upon a publication that critiqued the bioinformatics associated with Cap Analysis Gene Expression (CAGE) and developed novel methodologies to resolve repetitive element transcription. Chapter 3 describes the development of an updated CAGE mapping pipeline for the fourth stage of the international Functional Annotation of Mouse (FANTOM) project, which lead to the generation of a research article and a book chapter. These works demonstrated the enhanced utility of CAGE when coupled with next-generation sequencing, highlighted the benefits of CAGE when applied to systems biology and profiled the temporal expression of human repetitive elements. Chapter 4 presents an in-depth analysis of repetitive element transcription in the mouse and human genomes. Using CAGE, approximately 250,000 retrotransposon associated transcription start sites were defined, many of which were tissue-specific. Retrotransposons were found to frequently function as alternative promoters for protein-coding genes and/or express non-coding RNAs. Furthermore, when retrotransposons were found within the 3’UTR of protein-coding genes, there was strong evidence for the reduced expression of the corresponding transcripts. A genome-wide screen for strong expression correlation between repetitive elements and neighbouring protein-coding genes identified approximately 23,000 candidate regulatory regions derived from retrotransposons, including several hundred putative boundary elements. These were in addition to more than two thousand examples of bidirectional transcription found in retrotransposons, which are known to be a source of double stranded RNAs involved in RNA interference. Chapter 5 explores the proportion of the mouse embryonic stem cell transcriptome comprised of repeat-derived transcripts, using next-generation RNA sequencing. This study defined the dynamic expression of repetitive elements at the greatest resolution achieved to date and demonstrated that repetitive elements are an intrinsic part of the mammalian transcriptional landscape.
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Estudo de genes expressos em frutos de camu-camu: seqüenciamento de ests.Silva, Marcicleide Lima da 22 June 2006 (has links)
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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.
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Functional studies on WDR36 gene and its regulations in early male chicken embryogenesisLin, Yuan-Ping 08 September 2010 (has links)
From the sexual preselection point of view, understanding sex determination/differentiation mechanisms in the bird is critical in both evolutionary and industrial applications. The chicken embryo provides an unique vertebrate model in the field of development biology. Morphological sex development in the chick gonad starts at 6.5 embryonic day (E6.5), however, genetic sex determination and development should occur earlier. In order to comprehend genes and their underlying mechanisms being involved in sex-determination/development during early embryogenesis, we not only made a male-subtract-female and a female-subtract-male cDNA library as early as embryonic day 3 (E3; Hamburger and Hamilton Stage 20), but also examined early transcripts related to male development in chicken embryo and their expression profiles in this study. A total of 89 and 127 candidates of male-development transcripts represented respectively for 83 known and 119 unknown non-redundant sequences, which were characterized in an E3 male- subtract-female complementary DNA library. In this study, thirty-five selected transcripts being validated by quantitative reverse transcription-polymerase chain reaction that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). Notably, twelve of these transcripts mapped to the Z chromosome. At 72 weeks of age, twenty transcripts were expressed at higher levels in testes than in ovaries. Meanwhile, four transcripts were expressed at higher levels in brains of male than in brains of female chickens (P < 0.05). By using of methods of whole mount and frozen cross-section in situ hybridization, the expressions of riboflavin kinase (RFK), WD repeat domain 36 (WDR36) and EY505808 transcripts on E7 chicken male gonads were corroborated to be better than female gonads. This result was confirmed by using of western blotting analysis which also showed the expressions were specifically on gonads than other tissues. Treatment with an aromatase inhibitor formestane at E4 depicted the effect of the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), which was similar to the observed gene expressions for both doublesex and mab-3 related transcription factor 1 gene. Additionally, the interaction effects of sex with formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). This study demonstrated a panel of potentially candidate male development transcripts being identified during early chicken embryogenesis; some might be regulated by sex hormones.
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Studies on the protein expression of thermosensitive/Neural development-related gene in tilapia, Oreochromis mossambicus.Lu, Yu-nuo 27 January 2010 (has links)
Expressed sequence tags (ESTs) are derived from the developing
tilapia brain was established in our lab. There are 9 transcripts were
identified as thermosensitive/Neural development-related gene. The
effects of different temperatures on the ontogenetic expression of
these thermosensitive/Neural development-related gene during the
critical period of brain sexual differentiation were investigated in the
present study. The ontogenetic expression of inhibitor of DNA
binding/differentiation protein 2 (Id2), thermosensitive/Neural
development-related gene, were enhanced by both lower (20¢J) and
higher (32¢J) temperatures before 10 days post-hatching.
In this study, bioinformatics were searched for Id2, which is a
gene with 738 bp of patial cDNA sequence, open reading frame
(ORF) is 411bp, and deduced 137 amino acids of protein sequence.
The protein of Id2 was expressed in a prokaryotic system, BL21
(Escherichia coli) and purified with Ni-NTA affinity chromatography.
Also, the ORF of Id2 was cloned into pEGFP vector, and
plasmid (pEGFP-Id2) was transfected into the eukaryotic system,
mouse neuroblastoma cell (Neuro-2a cell). The distribution of Id2
expressed in the Neuro-2a cell was identified by fluorescence
microscopy.
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Development of gene-linked molecular markers in South African abalone (Haliotis midae) using an in silico mining approachRhode, Clint 03 1900 (has links)
Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The South African abalone, Haliotis midae, is the only endemic species of commercial value. Aquaculture remains the only avenue for expanding the industry, since the closure of the fishery. The current focus is on implementing a molecular breeding programme; thus the development of molecular markers for linkage mapping and QTL analysis is a priority. Various markers, mainly anonymous, have been developed for H. midae; however emphasis is being placed on the development of gene-linked type I molecular markers. The present study investigates and demonstrates the use of public sequence collections to develop type I markers for a species with limited genomic resources, via three strategies: Surveying anonymous H. midae microsatellite markers’ flanking regions to find homology to gene sequences in public databases, cross-species marker transfer of anonymous markers from H. rubra and H. discus hannai demonstrating putative gene associations and lastly EST marker mining (SNP and microsatellites) from various Haliotids and testing transfer to the target species. Approximately 17% of H. midae anonymous markers showed significant similarity to genes. The current study also reports higher cross-species transferability from both H. rubra and H. discus hannai to H. midae (39% and 20.5%, respectively) than previously demonstrated and 15 EST-microsatellites and 16 EST-SNPs were successfully mined. Furthermore, the non-random distribution of microsatellites and high nucleotide diversity in the H. midae genome was confirmed. This is a low cost and time effective method for marker development and presents a continuous and dynamic resource that could be used for future marker development and characterisation as sequence information in public databases grow exponentially. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse perlemoen, Haliotis midae, is die enigste van vyf inheemse spesies van kommersiële waarde. Na die noodgedwonge sluiting van die vissery, is akwakultuur die mees praktiese oplossing om die perlemoen industrie uit te brei. Die huidige fokus is gerig op die implementering van ‘n molekulêre teel-program en dus is die ontwikkeling van molekulêre merkers vir genetiese kartering en kwantitatiewe kenmerk lokus analise, van uiterste belang. Tipe II merkers is voorheen vir die perlemoen ontwikkel, maar huidige tendense lê klem op die ontwikkeling van geen-gekoppelde tipe I merkers. Die huidige studie ondersoek die gebruik van publieke databasisse vir die ontwikkeling van tipe I molekulêre merkers vir ‘n spesie met beperkte genomiese bronne. Drie strategieë is geïmplementeer: Eerstens is ‘n opname gemaak van die homologie van perlemoen tipe II merker-vleuelende volgordes met geen volgordes in databasisse. Verder is die oordraagbaarheid van tipe II merkers vanaf H. rubra en H. discus hannai wat assosiasie met gene toon ondersoek. Laastens is ‘n Uitgedrukte Volgorde Merk (UVM) (Expressed Sequence Tag, EST) merker-ontginnings metode vanaf verskeie Haliotis spesies en toetsing van oordraagbaarheid na die teiken spesie uitgevoer. Ongeveer 17% van die tipe II H. midae merkers het geniese assosiasie getoon. ‘n Hoër tussen-spesie oordraagbaarheid vanaf beide H. rubra en H. discus hannai na H. midae (39% en 20.5%, onderskeidelik) word gerapporteer in vergelyking met vorige studies en 15 UVM-mikrosatelliete en 16 UVM-enkel nukleotied polimorfismes (single nucleotide polimorphism, SNP) is ontwikkel. Verder bevestig die studie die nie-lukrake verspreiding van mikrosatelliete en hoë nukleotied diversiteit in die perlemoen genoom. Die gebruik van publieke databasise vir die ontwikkeling en karakterisering van tipe I molekulêre merkers is tyd- en koste-besparend en bied ‘n volgehoue en dinamiese bron vir toekomstige gebruik.
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Expressão gênica diferencial em palmitos de cana-de-açúcar submetida a diferentes períodos de estresse hídrico /Jovino, Daniele Fernanda Revoredo. January 2007 (has links)
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
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Molecular characterization of digestive proteases of the yellow mealworm, Tenebrio molitor L.Prabhakar, Sheila January 1900 (has links)
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.
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